U.S. patent application number 17/311574 was filed with the patent office on 2022-01-27 for collagen solid, method for producing collagen solid, biomaterial, and ex vivo material.
The applicant listed for this patent is KINKI UNIVERSITY. Invention is credited to Naomasa Fukase, Saori Kunii, Ryosuke Kuroda, Koichi Morimoto, Toshiyuki Takemori.
Application Number | 20220023499 17/311574 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-27 |
United States Patent
Application |
20220023499 |
Kind Code |
A1 |
Morimoto; Koichi ; et
al. |
January 27, 2022 |
COLLAGEN SOLID, METHOD FOR PRODUCING COLLAGEN SOLID, BIOMATERIAL,
AND EX VIVO MATERIAL
Abstract
The present invention provides a collagen solid having higher
strength and density. A collagen solid is used which contains a
collagen-cysteine protease degradation product or an
atelocollagen-cysteine protease degradation product and has a
density of 50 mg/cm.sup.3 or more.
Inventors: |
Morimoto; Koichi;
(Kinokawa-shi, JP) ; Kunii; Saori; (Kinokawa-shi,
JP) ; Fukase; Naomasa; (Kobe-shi, JP) ;
Kuroda; Ryosuke; (Kobe-shi, JP) ; Takemori;
Toshiyuki; (Kobe-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KINKI UNIVERSITY |
Higashiosaka-shi, Osaka |
|
JP |
|
|
Appl. No.: |
17/311574 |
Filed: |
December 12, 2019 |
PCT Filed: |
December 12, 2019 |
PCT NO: |
PCT/JP2019/048788 |
371 Date: |
June 7, 2021 |
International
Class: |
A61L 27/24 20060101
A61L027/24; C07K 14/78 20060101 C07K014/78 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 12, 2018 |
JP |
2018-232811 |
Apr 22, 2019 |
JP |
2019-081130 |
Claims
1. A collagen solid, comprising a collagen-cysteine protease
degradation product or an atelocollagen-cysteine protease
degradation product, wherein: said collagen solid has a density of
50 mg/cm.sup.3 or more; each of the collagen-cysteine protease
degradation product and the atelocollagen-cysteine protease
degradation product includes a triple helical domain of collagen;
the triple helical domain includes at least 100 amino acid
sequences represented by Gly-X-Y (where each of X and Y is any
amino acid); and in each of the collagen-cysteine protease
degradation product and the atelocollagen-cysteine protease
degradation product, three polypeptide chains form a helical
structure.
2. The collagen solid as set forth in claim 1, wherein said
collagen solid has a tangent modulus of 90 kPa or more.
3. The collagen solid as set forth in claim 1, further comprising
an optional substance.
4. A biomaterial, comprising the collagen solid of claim 1.
5. An ex vivo material, comprising the collagen solid of claim
1.
6. A bone regeneration material, comprising the collagen solid of
claim 1.
7. A method for producing the collagen solid of claim 1, said
method comprising: a degradation step of degrading collagen or
atelocollagen with a cysteine protease; and a removal step of
removing a solvent from a collagen degradation product or an
atelocollagen degradation product which has been obtained in the
degradation step.
8. The method as set forth in claim 7, wherein, in the removal
step, an optional substance is added to the collagen degradation
product or the atelocollagen degradation product which has been
obtained in the degradation step to obtain a mixture, and then the
solvent is removed from the mixture.
9. The method as set forth in claim 7, wherein, in the removal
step, a collagen solid which has been obtained in the removal step
is caused to adsorb an optional substance.
10. The collagen solid of claim 1, wherein: said collagen solid has
a disk shape, a tube shape, a columnar shape, a conical shape, an
arrowhead shape, a hexahedral shape, a polyhedral shape, a
polygonal column shape, a bellows shape, a screw shape, a male
screw shape, a female screw shape, or a shape in which two or more
of these shapes are connected to each other, or said collagen solid
is in a form of powder.
Description
TECHNICAL FIELD
[0001] The present invention relates to a collagen solid, a method
for producing the collagen solid, a biomaterial, and an ex vivo
material.
BACKGROUND ART
[0002] Collagen, which is a protein constituting a connective
tissue between cells or a bone tissue of an animal, or gelatin,
which is a thermally denatured material of collagen, has been
conventionally used in various applications.
[0003] If a bone is defective, complete self-renewal is difficult.
Therefore, bone regeneration therapy using an autologous bone or a
bone derived from a conspecific organism has been conventionally
carried out. For transplantation of an autologous bone, it is
necessary to extract a portion of one's own bone, and there is a
limit to an amount of bone that can be extracted from our body.
Meanwhile, for transplantation of a bone derived from a conspecific
organism, a bone of another person is used. Therefore, a risk of
infection is high. Under the circumstances, in recent years, bone
regeneration therapy has been carried out using biomaterials such
as hydroxyapatite or .beta.-TCP. However, the biomaterials
themselves described above do not have an ability to create a new
bone, and a rate of bone formation is significantly inferior to
that of an autologous bone. In recent years, bone regeneration
therapy has been carried out using stem cells and scaffolds formed
of biomaterials. However, there are many problems in this
technique, such as the need for a cell culture facility and a high
cost. Under such circumstances, development of a new technique for
bone regeneration therapy is currently demanded.
[0004] Collagen, which is a protein constituting a connective
tissue between cells or a bone tissue of an animal, is
conventionally used as a biomaterial to be implanted in a living
body (e.g., as a biomaterial to complement a defective or damaged
biological tissue).
[0005] Meanwhile, collagen can form a scaffold for cells to adhere
to a substrate. Therefore, conventionally, a technique of forming a
scaffold by coating a substrate with an aqueous solution containing
collagen has been used.
[0006] For example, Patent Literature 1 discloses a composite type
of bone filling material composed of a combination of collagen and
a calcium phosphate compound. Patent Literature 2 discloses a
biomaterial containing collagen, calcium phosphate and sugar as
main components. Non-patent Literature 1 discloses a biomaterial
composed of collagen crosslinked by glutaraldehyde.
[0007] In addition, Patent Literature 3 discloses a degradation
product of collagen or atelocollagen, a method for producing the
degradation product, and use of the degradation product. The
degradation product has spheroid-forming activity. Meanwhile.
Patent Literature 4 discloses a differentiation-inducing
composition containing a degradation product of collagen or
atelocollagen. The differentiation-inducing composition has
spheroid-forming activity, bone differentiation inducing ability,
and the like.
[0008] In addition, in the field of carrying out cell culture,
coating a substrate with an aqueous solution containing collagen is
widely conducted.
CITATION LIST
Patent Literature
[0009] [Patent Literature 1] [0010] Japanese Patent Application
Publication Tokukai No. 2000-262608 [0011] [Patent Literature 2]
[0012] Japanese Patent Application Publication Tokukai No.
2009-5814 [0013] [Patent Literature 3] [0014] WO2015/167003 [0015]
[Patent Literature 4] [0016] WO2015/167004
Non-Patent Literature
[0016] [0017] [Non-patent Literature 1] [0018] L. H. H. Olde Demink
et. al., Journal of Materials science: Materials in Medicine: 6,
pp. 460-472, 1995
SUMMARY OF INVENTION
Technical Problem
[0019] Sites in a living body where a biomaterial is to be
implanted often have complex shapes. In order for the biomaterial
to function well in the living body, it is important to adapt a
shape of the biomaterial to a shape of a site at which the
biomaterial is to be implanted. However, the conventional
biomaterial has a problem that the biomaterial does not have high
strength (in other words, the conventional biomaterial is soft) and
therefore cannot be processed into an intended shape.
[0020] Moreover, the technique of coating a substrate with an
aqueous solution containing collagen has a problem that a high
concentration of collagen, which is comparable to a collagen
concentration in a living body, cannot be adsorbed on a substrate
surface.
[0021] The degradation product disclosed in Patent Literature 3 is
in the form of solution. Patent Literature 3 does not disclose a
concept of concentrating the degradation product into a highly
dense solid. Of course, Patent Literature 3 does not disclose a
tangent modulus of the solid. Further, the differentiation-inducing
composition disclosed in Patent Literature 4 is also in the form of
solution. Patent Literature 4 does not disclose a concept of
concentrating the degradation product into a highly dense solid. Of
course, Patent Literature 4 does not disclose a tangent modulus of
the solid.
[0022] As described above, both of the degradation product
disclosed in Patent Literature 3 and the differentiation-inducing
composition disclosed in Patent Literature 4 are in the form of
solution. The degradation product and the differentiation-inducing
composition which are implanted into living bodies easily diffuse
and are lost from the implanted sites. As such, there has been room
for further improvement in regeneration of a living tissue (e.g.,
bone) at a target site. Moreover, even if the substrate is coated
with the degradation product or the differentiation-inducing
composition, it is not possible to adsorb a high concentration of
collagen, which is comparable to the collagen concentration in a
living body, on the substrate surface. There has been room for
further improvement in adsorption of a high concentration of
collagen, which is comparable to the collagen concentration in a
living body, on the substrate surface.
[0023] In addition, a bone (e.g., femur) is a biological tissue to
which a large load is to be applied and, when a biomaterial is
implanted into a bone or the like, it is demanded that the
biomaterial itself has high strength. Moreover, there is a
possibility that a large load is to be applied to a substrate or
the like used in cell culture, and/or the substrate is to be used
in long-term culture. Therefore, the substrate or the like needs to
have high strength. However, the conventional material has a
problem that the material does not have high strength (in other
words, the conventional material is soft).
[0024] In order to achieve high strength in a conventional
material, it is necessary to employ an auxiliary material different
from collagen (e.g., a crosslinking agent or a synthetic polymer).
However, the auxiliary material has the following problems: (i) the
auxiliary material increases a cost of material and/or increases
immunogenicity, inflammation, retention, and the like of the
material in a living body, thereby reducing safety; and (ii) the
auxiliary material adversely affects cells to be cultured and/or
makes it impossible to culture cells under the same conditions as
in the living body.
[0025] In addition, conventional materials have a problem that it
is difficult to incorporate an optional substance component into
the material because, for example, solubility of the raw material
is low or strength of the material is low.
[0026] An object of the present invention is to provide a collagen
solid which has a high density and high strength.
Solution to Problem
[0027] In a case where collagen is degraded using a protease such
as pepsin, merely a solution is obtained which contains a large
amount of insoluble precipitate and soluble impurities and contains
a soluble degradation product at a low concentration (i.e., 20
mg/mL or less as shown in Example described later).
[0028] The inventors of the present invention have found the
following facts: (i) by degrading collagen or atelocollagen using a
cysteine protease, it is possible to obtain a solution containing a
solubilized degradation product at a high concentration (i.e., 30
mg/mL or more as shown in Example described later): (ii) by
removing a solvent from the solution, it is possible to obtain a
collagen solid (hereinafter referred to as "LASCol", which is an
abbreviation of low adhesive scaffold collagen) which contains the
collagen degradation product at a high density (i.e., 50
mg/cm.sup.3 or more as shown in Example described later), and/or
has a large tangent modulus (i.e., 90 kPa or more as shown in
Example described later), and consequently can be processed into an
intended shape; and (iii) the collagen solid (LASCol) is prepared
from collagen that is originally present in a living body, and
therefore can be used safely and reproducibly as a biomaterial for
bone regeneration and a biomaterial for cell culture. On the basis
of those findings, the inventors have accomplished the present
invention.
[1] In order to attain the object, a collagen solid in accordance
with an aspect of the present invention contains a
collagen-cysteine protease degradation product or an
atelocollagen-cysteine protease degradation product, the collagen
solid having a density of 50 mg/cm.sup.3 or more. [2] The collagen
solid in accordance with an aspect of the present invention for
attaining the object has a tangent modulus of 90 kPa or more. [3]
The collagen solid in accordance with an aspect of the present
invention further contains an optional substance. [4] In order to
attain the object, a biomaterial in accordance with an aspect of
the present invention contains the collagen solid described in any
one of [1] through [3]. [5] In order to attain the object, an ex
vivo material in accordance with an aspect of the present invention
contains the collagen solid described in any one of [1] through
[3]. [6] In order to attain the object, a biomaterial in accordance
with an aspect of the present invention is a bone regeneration
material containing the collagen solid described in any one of [1]
through [3]. [7] In order to attain the object, a method for
producing a collagen solid in accordance with an aspect of the
present invention is a method for producing the collagen solid
described in any one of [1] through [3] and includes: a degradation
step of degrading collagen or atelocollagen with a cysteine
protease; and a removal step of removing a solvent from a collagen
degradation product or an atelocollagen degradation product which
has been obtained in the degradation step. [8] In the method for
producing a collagen solid in accordance with an aspect of the
present invention, in the removal step, an optional substance is
added to the collagen degradation product or the atelocollagen
degradation product which has been obtained in the degradation step
to obtain a mixture, and then the solvent is removed from the
mixture. [9] In the method for producing a collagen solid in
accordance with an aspect of the present invention, in the removal
step, a collagen solid which has been obtained in the removal step
is caused to adsorb an optional substance.
Advantageous Effects of Invention
[0029] According to an aspect of the present invention, it is
possible to provide a collagen solid having a higher density and
higher strength. Further, according to an aspect of the present
invention, it is possible to provide a collagen solid which can be
easily processed into an intended shape. Further, according to an
aspect of the present invention, it is possible to provide a novel
biomaterial or ex vivo material containing a collagen solid having
a higher density and higher strength. Further, according to an
aspect of the present invention, it is possible to provide a
collagen solid which contains one or more optional substances in an
arbitrary amount, and to provide a novel biomaterial or ex vivo
material containing such a collagen solid. Further, according to an
aspect of the present invention, it is possible to provide a
biomaterial (in other words, bone regeneration material) which can
regenerate or repair a biological tissue (e.g., bone). Further,
according to an aspect of the present invention, it is possible to
provide an ex vivo material (in other words, material for cell
culture) which can be used in cell culture.
BRIEF DESCRIPTION OF DRAWINGS
[0030] (a) through (c) of FIG. 1 show cross-sectional images of
columnar collagen solids in accordance with Example of the present
invention.
[0031] (d) through (f) of FIG. 2 show cross-sectional images of
columnar collagen solids in accordance with Example of the present
invention.
[0032] (a) and (b) of FIG. 3 show cross-sectional images of
columnar atelocollagen solids in accordance with Comparative
Example of the present invention.
[0033] (a) of FIG. 4 is an image showing a bellows-shaped collagen
solid in a bent state, and (b) of FIG. 4 is an image showing the
bellows-shaped collagen solid in a stretched state.
[0034] (a) through (c) of FIG. 5 show images of
small-diameter-tubular collagen solids in accordance with Example
of the present invention.
[0035] (a) through (f) of FIG. 6 show SEM images and results of
SEM-EDX analysis of columnar collagen solids in accordance with
Examples of the present invention.
[0036] (a) and (b) of FIG. 7 show images of a columnar collagen
solid having small holes in accordance with Example of the present
invention.
[0037] (a) through (h) of FIG. 8 show SEM images and results of
SEM-EDX analysis of collagen solids in accordance with Example of
the present invention.
[0038] (a) and (b) of FIG. 9 are images showing processes for
preparing a 4 mm femur defect rat model in accordance with Example
of the present invention.
[0039] FIG. 10 is a view showing evaluation criteria of the
modified RUST score used in evaluation with medical imaging
technology in accordance with Example of the present invention.
[0040] FIG. 11 shows radiographic images of bones of a 1 mm femur
defect population, a 50 mg/mL LASCol solid implanted population, a
100 mg/mL LASCol solid implanted population, and a 150 mg/mL LASCol
solid implanted population taken immediately after implantation, 14
days after implantation, and 28 days after implantation, in
accordance with Example of the present invention.
[0041] FIG. 12 is a graph showing results of evaluating bone
adhesion on the 28th day after implantation based on the modified
RUST score in the 1 mm femur defect population, the 50 mg/mL LASCol
solid implanted population, the 100 mg/mL LASCol solid implanted
population, and the 150 mg/mL LASCol solid implanted population in
accordance with Example of the present invention.
[0042] FIG. 13 shows .mu.CT images of the 1 mm femur defect
population, the 50 mg/mL LASCol solid implanted population, the 100
mg/mL LASCol solid implanted population, and the 150 mg/mL LASCol
solid implanted population taken 28 days after implantation, in
accordance with Example of the present invention.
[0043] FIG. 14 shows .mu.CT images of the 100 mg/mL LASCol solid
implanted population taken 28 days after implantation, in
accordance with Example of the present invention.
[0044] FIG. 15 shows .mu.CT images of the 150 mg/mL LASCol solid
implanted population taken 14 days after implantation, in
accordance with Example of the present invention.
[0045] FIG. 16 shows .mu.CT images of the 150 mg/mL LASCol solid
implanted population taken 28 days after implantation, in
accordance with Example of the present invention.
[0046] FIG. 17 is an image showing a femur extracted from a rat in
the 150 mg/mL LASCol solid implanted population taken 28 days after
implantation, in accordance with Example of the present
invention.
[0047] FIG. 18 is a view showing histological evaluation criteria
based on the Allen's score, in accordance with Example of the
present invention.
[0048] FIG. 19 shows HE stained images of sectioned femur tissues
of a 1 mm femur defect individual and a 50 mg/mL LASCol solid
implanted individual taken 14 days after implantation and 28 days
after implantation, in accordance with Example of the present
invention.
[0049] FIG. 20 shows SO stained images of the same sectioned
tissues as those in FIG. 19 (i.e., the sectioned femur tissues of a
1 mm femur defect individual and a 50 mg/mL LASCol solid implanted
individual on the 14th day after implantation and 28th day after
implantation) and evaluation results based on the Allen's score, in
accordance with Example of the present invention.
[0050] FIG. 21 shows SO stained images of sectioned femur tissues
of a 100 mg/mL LASCol solid implanted population taken 28 days
after implantation and evaluation results based on the Allen's
score, in accordance with Example of the present invention.
[0051] FIG. 22 shows SO stained images of sectioned femur tissues
of a 150 mg/mL LASCol solid implanted population taken 28 days
after implantation, in accordance with Example of the present
invention.
[0052] FIG. 23 is a graph showing results of evaluating sectioned
femur tissues 28 days after implantation based on the Allen's score
in the 1 mm femur defect population, the 50 mg/mL LASCol solid
implanted population, the 100 mg/mL LASCol solid implanted
population, and the 150 mg/mL LASCol solid implanted population in
accordance with Example of the present invention.
[0053] FIG. 24 shows a .mu.CT image of a CaCO.sub.3-containing 150
mg/mL LASCol solid implanted individual taken 14 days after
implantation, in accordance with Example of the present
invention.
[0054] (a) of FIG. 25 shows a .mu.CT image of a bFGF-containing 100
mg/mL LASCol solid implanted individual taken 35 days after
implantation, in accordance with Example of the present invention.
(b) of FIG. 25 shows a .mu.CT image of a 4 mm femur defect
individual taken 35 days after implantation, in accordance with
Example of the present invention.
DESCRIPTION OF EMBODIMENTS
[0055] The following description will discuss an embodiment of the
present invention. The present invention is, however, not limited
to the embodiment below. The present invention is not limited to
arrangements described below, but may be altered in various ways by
a skilled person within the scope of the claims. The present
invention also encompasses, in its technical scope, any embodiment
and any working example derived by appropriately combining
technical means disclosed in differing embodiments. Moreover, all
literatures described in this specification are incorporated herein
as reference literatures. Any numerical range "A to B" expressed in
the present specification intends to mean "not less than A and not
more than B".
[0056] [1. Conception by Inventors]
[0057] Collagen, which is a protein constituting a connective
tissue between cells or a bone tissue of an animal, or gelatin,
which is a thermally denatured material of collagen, can be used as
a biomaterial to be implanted in a living body (e.g., as a
biomaterial to complement a defective or damaged biological tissue)
or as an ex vivo material (e.g., an ex vivo material for cell
culture).
[0058] Collagen and atelocollagen are poorly soluble in water.
Therefore, in a case where a biomaterial or an ex vivo material is
prepared using an aqueous solution in which collagen or
atelocollagen is dissolved, only a solution-state substance (gel)
with a low concentration or a solid-state substance (sponge) with a
low density can be prepared. The solution-state substance has a
maximum concentration of approximately 20 mg/mL, and the
solid-state substance has a maximum density of approximately 26
mg/cm.sup.3. Such a low-concentration or low-density collagen
preparation has a disadvantage of extremely low mechanical strength
as a biomaterial or an ex vivo material. In addition, the density
of collagen present in living organisms is 200 mg/cm.sup.3 or more,
and conventional collagen preparations can reproduce only
approximately 1/10 of that density.
[0059] In order to overcome the disadvantage, a method may be
employed which increases the mechanical strength by disorderly
crosslinking collagen with collagen by heat, light, a chemical
substance, or the like. This method increases the mechanical
strength as a biomaterial or an ex vivo material but has a risk of
increasing antigenicity (immunogenicity) in a living body. Further,
in this method, even if a sponge-like solid having a large porosity
is crosslinked, it is difficult to reduce voids in the sponge-like
solid. Thus, although an elastic solid can be obtained by this
method, it is not possible to obtain hard solids having a high
density, such as wood blocks and metals. Therefore, conventionally,
there has been no idea per se to use a dense freeze-dried product
of collagen as a biomaterial or an ex vivo material.
[0060] Gelatin is more hydrophilic and more soluble in water, as
compared with collagen. Therefore, if a biomaterial or an ex vivo
material is prepared using an aqueous solution in which gelatin is
dissolved, a density of the gelatin preparation can be increased up
to 800 mg/cm.sup.3. Thus, the gelatin preparation can be prepared
as a solid-state substance having a high density. However, gelatin
has great solubility in water. Therefore, in a case where the
gelatin preparation is injected into a living body, the gelatin
preparation is immediately dissolved. In addition, gelatin is
rapidly degraded by endogenous peptidases. Therefore, in vivo
retention of the gelatin preparation is extremely low. It may also
be conceivable to crosslink gelatin with gelatin as with collagen
to control biodegradability of gelatin preparations. However, such
a method has a risk of increasing antigenicity (immunogenicity) in
a living body.
[0061] In other words, in collagen preparations and gelatin
preparations, it is technically difficult to collect a large amount
of the same crosslinked products by carrying out a highly
reproducible crosslinking treatment, and further, there are many
problems concerning in vivo safety of such crosslinked products. In
the conventional technique, only a mixture of crosslinked products
having different crosslinking numbers and crosslinking positions
and having various molecular sizes is obtained. In the conventional
technique, it is impossible to prepare a mixture of crosslinked
products having the same crosslinking number and crosslinking
position and having the same porosity.
[0062] In order to utilize collagen as a biomaterial or an ex vivo
material, the present inventors have attempted to develop a new
technique for producing a collagen preparation having a high
concentration or a high density without crosslinking. If a collagen
preparation having a high concentration or a high density
comparable to the collagen concentration or density in a living
body is realized, such a collagen preparation is expected to have
high mechanical characteristics which are not conventionally seen,
and is expected to be utilized not only in vivo but also in
vitro.
[0063] The collagen preparation having a high concentration or a
high density may be utilized in vivo as a biomaterial that allows
bone regeneration in a bone defect patient. Specifically, if a bone
is defective, complete self-renewal is difficult. Therefore, bone
regeneration therapy using an autologous bone or a bone derived
from a conspecific organism has been conventionally carried out.
For transplantation of an autologous bone, it is necessary to
extract a portion of one's own bone, and there is a limit to an
amount of bone that can be extracted. Meanwhile, for
transplantation of a bone derived from a conspecific organism, a
bone of another person is used. Therefore, a risk of infection is
high. It is also conceivable that bone regeneration therapy is
carried out using, as an artificial bone filler, a bone filler
composed of calcium phosphate such as hydroxyapatite or .beta.-TCP
as a base material. A mixture of calcium phosphate and collagen or
gelatin has been commercialized as a bone filler. In order to
increase affinity for cells in vivo, calcium phosphate and collagen
or gelatin can be mixed to form a bone filler. However, the bone
filler does not have sufficient mechanical strength as a bone
filler. Therefore, in the conventional technique, in order to
improve the mechanical strength, a ratio of calcium phosphate is
higher than that of collagen or gelatin. However, the bone filler
itself described above does not have an ability to create a new
bone, and a rate of bone formation is significantly inferior to
that of an autologous bone. In recent years, bone regeneration
therapy has been carried out using stem cells and scaffolds formed
of bone filler. However, there are many problems in this technique,
such as the need for a cell culture facility and a high cost. Under
the circumstances, the present inventors have attempted to develop
a new technique for bone regeneration therapy.
[0064] As a utilization method of collagen preparation having a
high concentration or a high density in vivo and in vitro, the
collagen preparation can be expected to be utilized as a
three-dimensional scaffold for cell culture. In a case where a
conventional commercially available collagen solution is used as a
scaffold, the collagen solution which has a low concentration of 3
mg/mL is applied on a substrate to form a scaffold, or the gelled
collagen solution is used as a scaffold. In this case, the
concentration of collagen in the scaffold is greatly different from
the in vivo environment, and it is therefore difficult to know
functions and behavior of original cells. For example, in a case
where primary cells taken from a tissue or the like are cultured on
a scaffold having a low concentration of collagen, the cell cycle
progresses faster, and cell growth is easily started. Although this
case is advantageous to increase the number of primary cells, such
extreme cell growth does not occur in a living body. Moreover, in
such an environment, primary cells are generally prone to
dedifferentiation. Once the primary cells are dedifferentiated, the
function of the primary cells is lost, and this makes it impossible
to investigate the function of the primary cells. In other words,
there are many problems in the technique of confirming the original
function of cells using conventional commercially available
collagen solutions. Therefore, it is urgently necessary to develop
a new scaffold imitating the in vivo environment in the field of
regenerative medical techniques, the field of cell biology, the
field of developmental biology, and the like.
[0065] For example, Patent Literature 1 discloses a composite type
of bone filling material composed of a combination of collagen and
a calcium phosphate compound. Patent Literature 2 discloses a
biomaterial containing collagen, calcium phosphate and sugar as
main components. Non-patent Literature 1 discloses a biomaterial
composed of collagen crosslinked by glutaraldehyde.
[0066] In addition, Patent Literature 3 discloses a degradation
product of collagen or atelocollagen, a method for producing the
degradation product, and use of the degradation product. The
degradation product has spheroid-forming activity. Meanwhile,
Patent Literature 4 discloses a differentiation-inducing
composition containing a degradation product of collagen or
atelocollagen. The differentiation-inducing composition has
spheroid-forming activity, bone differentiation inducing ability,
and the like.
[0067] Sites in a living body where a biomaterial is to be
implanted often have complex shapes. In order for the biomaterial
to function well in the living body, it is important to adapt a
shape of the biomaterial to a shape of a site at which the
biomaterial is to be implanted. However, the conventional
biomaterial and ex vivo material have a problem that the
biomaterial and the ex vivo material do not have a high density and
high strength (hardness) (in other words, the conventional
biomaterial and ex vivo material are soft) and therefore cannot be
processed into an intended shape. That is, the conventional
biomaterial and ex vivo material are sponge-like solids having a
high porosity, and high strength (hardness) cannot be given to such
biomaterial and ex vivo material. This problem is common to all of
known sponge-like solids prepared from protein components and
cannot be solved. Nobody could imagine making uniform solids having
few voids (like metal) with only a protein. If a solid having a
high density and high strength (hardness) can be prepared from a
single collagen, it will be possible to open up an entirely new
application for the solid. If a solid having a high density and
high strength (hardness) can be prepared from a single collagen, it
is possible to prepare a solid having an intended shape (e.g., a
cubic shape, a columnar shape, a disk shape, a straight tube shape,
a curved tube shape, a screw shape, a male screw shape, a female
screw shape, a film shape, and the like) by using an appropriate
template. Preparing such a shaped product using collagen originally
present in a living body is a breakthrough and the utility value of
such a shaped product is immeasurable.
[0068] The degradation product disclosed in Patent Literature 3 is
in the form of solution. Patent Literature 3 does not disclose a
concept of concentrating the degradation product into a highly
dense solid. Of course, Patent Literature 3 does not disclose a
tangent modulus of the solid. Further, the differentiation-inducing
composition disclosed in Patent Literature 4 is also in the form of
solution. Patent Literature 4 does not disclose a concept of
concentrating the degradation product into a highly dense solid. Of
course, Patent Literature 4 does not disclose a tangent modulus of
the solid.
[0069] Indeed, a solid having a high density is not known so far
which is prepared by freeze-drying an aqueous solution containing a
single undenatured protein at a concentration of 50 mg/mL or more.
If such a solid can be prepared, such a solid can be utilized as an
entirely new biomaterial or ex vivo material.
[0070] For example, both of the degradation product disclosed in
Patent Literature 3 and the differentiation-inducing composition
disclosed in Patent Literature 4 are in the form of solution or in
a low density state. The degradation product and
differentiation-inducing composition which are implanted into
living bodies easily diffuse and are lost from the implanted sites.
As such, there has been room for further improvement in
regeneration of a living tissue (e.g., bone) at a target site.
[0071] In addition, a bone (e.g., femur) is a biological tissue to
which a large load is to be applied and, when a biomaterial is
implanted into a bone or the like, it is demanded that the
biomaterial itself has high strength. However, the conventional
biomaterial has a problem that the biomaterial does not have high
strength (in other words, the conventional biomaterial is soft) and
therefore can be implanted into only limited biological
tissues.
[0072] Furthermore, conventionally, collagen has been widely used
as an ex vivo material, i.e., a scaffold for cell culture. However,
in the conventional technique, a collagen-containing aqueous
solution at a low concentration of approximately 3 mg/mL is applied
to a culture dish, and cells are cultured on the culture dish.
Alternatively, a collagen-containing aqueous solution having a low
concentration of approximately 3 mg/mL is gelled on a culture dish,
and then cells are cultured on the culture dish. However, the
concentration of collagen in vivo is not the low concentration of
approximately 3 mg/mL but is a high concentration. Therefore, the
conventional technique has a problem that cells cannot be cultured
in vitro in a condition similar to an in vivo condition. Further,
there is a problem that a shaped product having a shape (e.g., a
cube) suitable for culture cannot be prepared.
[0073] In order to achieve high strength in a conventional
biomaterial or ex vivo material, it is necessary to employ an
auxiliary material different from collagen (e.g., a crosslinking
agent or a synthetic polymer). However, the auxiliary material has
a problem that the auxiliary material increases a cost of
biomaterial and/or increases immunogenicity, inflammation,
retention, and the like of the biomaterial in a living body,
thereby reducing safety. Further, even if a large amount of
auxiliary material is added to the biomaterial or ex vivo material
to increase the strength of the biomaterial or ex vivo material,
the density of collagen itself contained in the biomaterial or ex
vivo material cannot be increased. That is, there is a problem that
the density of collagen present in a living body cannot be
reproduced using conventional collagen. In other words, in the
conventional technique, although it is possible to prepare a
sponge-like solid which is reinforced in strength and has a high
porosity from a collagen preparation or a gelatin preparation, it
has been impossible to prepare a uniform collagen solid or gelatin
solid having little porosity like metals. It is conceivable to
prepare a highly dense solid by compressing the sponge-like solid.
However, when the compressive force is released, the solid expands
back to the original sponge-like solid. In order to obtain a highly
dense solid, it is also conceivable to crosslink the sponge-like
solid in a compressed state. However, such a crosslinked solid is
an artifact that does not exist in nature.
[0074] In addition, conventional biomaterials or ex vivo materials
have a problem that it is difficult to incorporate an optional
substance component into the biomaterial or ex vivo material
because, for example, solubility of the raw material is low or
strength of the biomaterial or ex vivo material is low.
[0075] An object of the present invention is to provide a collagen
solid having a high density and high strength (in other words, a
shaped product which is close to an in vivo environment and has a
low porosity).
[0076] [2. Method for Producing Collagen Solid]
[0077] The method in accordance with an embodiment of the present
invention for producing a collagen solid includes (i) a degradation
step of degrading collagen or atelocollagen with a cysteine
protease (specifically, partially cutting both ends of collagen or
atelocollagen with the cysteine protease) and (ii) a removal step
of removing a solvent from a collagen degradation product or an
atelocollagen degradation product which has been obtained in the
degradation step. The following description will discuss those
steps.
[0078] [2-1. Degradation Step]
[0079] In the degradation step, collagen or atelocollagen is
degraded with a cysteine protease.
[0080] The collagen is not limited to any particular one, and may
be any well-known collagen. Examples of the collagen include
collagens of (i) mammals (for example, a cow, a pig, a rabbit, a
human, a rat, and a mouse), (ii) birds (for example, a chicken), or
(iii) fishes (for example, a shark, a carp, an eel, a tuna [for
example, a yellowfin tuna], a tilapia, a sea bream, and a
salmon).
[0081] Further specifically, examples of the collagen include (i)
collagen derived from, for example, a dermis, a tendon, a bone, or
a fascia of any of mammals or birds and (ii) collagen derived from,
for example, a skin or a scale of any of fishes.
[0082] Examples of the atelocollagen include atelocollagen which is
produced by treating collagen of any of mammals, birds, or fishes
with a protease (for example, pepsin) and in which a telopeptide(s)
has been partially removed from the amino terminus and/or carboxyl
terminus of the collagen molecules.
[0083] A preferable option among the above examples is collagen or
atelocollagen of a chicken, a pig, a human, or a rat. A further
preferable option among the above examples is collagen or
atelocollagen of a pig or a human.
[0084] Collagen or atelocollagen of a fish can be prepared safely
in a large amount, and it is possible to provide a collagen solid
that is safer with respect to humans.
[0085] In a case where collagen or atelocollagen of a fish is used,
(i) a preferable option is collagen or atelocollagen of a shark, a
carp, an eel, a tuna (for example, a yellowfin tuna), a tilapia, a
sea bream, or a salmon, and (ii) a further preferable option is
collagen or atelocollagen of a tuna, a tilapia, a sea bream, or a
salmon.
[0086] The collagen may be prepared by a well-known method. For
example, collagen-rich tissue of a mammal, a bird, or a fish is put
into an acid solution with a pH of approximately 2 to 4 for elution
of collagen. Further, a protease such as pepsin is added to the
eluate for partial removal of a telopeptide(s) at the amino
terminus and/or carboxyl terminus of the collagen molecules. Then,
a salt such as sodium chloride is added to the eluate to
precipitate atelocollagen.
[0087] In a case where atelocollagen is used, the atelocollagen has
a heat denaturation temperature of preferably not lower than
15.degree. C. more preferably not lower than 20.degree. C. In a
case where, for example, atelocollagen of a fish is used, the
atelocollagen is preferably derived from a tuna (for example, a
yellowfin tuna), a tilapia, a carp, or the like because such
atelocollagen has a heat denaturation temperature of not lower than
25.degree. C. The above feature allows for production of a collagen
solid that is excellent in stability in storage and in use.
[0088] The cysteine protease is preferably (i) a cysteine protease
that contains a larger amount of acidic amino acids than that of
basic amino acids or (ii) a cysteine protease that is active at a
hydrogen ion concentration in an acidic region.
[0089] Examples of such a cysteine protease include cathepsin B [EC
3.4.22.1], papain [EC 3.4.22.2], ficin [EC 3.4.22.3], actinidain
[EC 3.4.22.14], cathepsin L [EC 3.4.22.15], cathepsin H [EC
3.4.22.16], cathepsin S [EC 3.4.22.27], bromelain [EC 3.4.22.32],
cathepsin K [EC 3.4.22.38], alloline, and calcium dependent
protease.
[0090] Among those, it is preferable to use papain, ficin,
actinidain, cathepsin K, alloline, or bromelain, and it is further
preferable to use papain, ficin, actinidain, or cathepsin K.
[0091] The enzyme can be prepared by a publicly known method.
Examples of such a method include (i) a method of preparing an
enzyme by chemical synthesis; (ii) a method of extracting an enzyme
from a bacterium, a fungus, or a cell or tissue of any of various
animals and plants; and (iii) a method of preparing an enzyme by a
genetic engineering means. The enzyme can alternatively be a
commercially available enzyme as well.
[0092] In a case where collagen or atelocollagen is degraded with
use of an enzyme (specifically, a cysteine protease), the
degradation can be carried out by, for example, any of the methods
(i) through (iii) below. The methods (i) through (iii) below are,
however, mere examples, and the present invention is not limited to
the methods (i) through (iii).
[0093] The methods (i) and (ii) below are each an example method
for cleaving a chemical bond at a particular position in the amino
acid sequence in (1) or (2) described later, and the method (iii)
below is an example method for cleaving a chemical bond at a
particular position in the amino acid sequence in (3) described
later.
(i) Method of causing collagen or atelocollagen to be in contact
with an enzyme in the presence of a salt having a high
concentration. (ii) Method of causing collagen or atelocollagen to
be in contact with an enzyme having been in contact with a salt
having a high concentration. (iii) Method of causing collagen or
atelocollagen to be in contact with an enzyme in the presence of a
salt having a low concentration.
[0094] A specific example of the method (i) above is a method of
causing collagen or atelocollagen to be in contact with an enzyme
in an aqueous solution containing a salt at a high
concentration.
[0095] A specific example of the method (ii) above is a method of
causing an enzyme to be in contact in advance with an aqueous
solution containing a salt at a high concentration and then causing
collagen or atelocollagen to be in contact with that enzyme.
[0096] A specific example of the method (iii) above is a method of
causing collagen or atelocollagen to be in contact with an enzyme
in an aqueous solution containing a salt at a low
concentration.
[0097] The aqueous solution is not particularly limited in terms of
specific arrangements. The aqueous solution can, for example,
contain water as a solvent.
[0098] The salt is not particularly limited in terms of specific
arrangements, but is preferably a chloride. The chloride is not
limited to any particular one. Examples of the chloride include
NaCl, KCl, LiCl, and MgCl.sub.2.
[0099] The salt contained in the aqueous solution at a high
concentration may have any concentration. A higher concentration
is, however, more preferable. The concentration is, for example,
preferably not less than 200 mM, more preferably not less than 500
mM, even more preferably not less than 1000 mM, even more
preferably not less than 1500 mM, most preferably not less than
2000 mM.
[0100] The concentration of the salt contained in the aqueous
solution at a high concentration may have any upper limit. The
upper limit may be 2500 mM, for example. A salt concentration of
higher than 2500 mM will salt out a large amount of protein, with
the result that the enzymatic degradation of collagen or
atelocollagen tends to have a decreased efficiency. A salt
concentration of not more than 2500 mM allows for a higher
efficiency of enzymatic degradation of collagen or
atelocollagen.
[0101] It follows that the concentration of the salt contained in
the aqueous solution at a high concentration is preferably within a
range of not less than 200 mM and not more than 2500 mM, more
preferably within a range of not less than 500 mM and not more than
2500 mM, even more preferably within a range of not less than 1000
mM and not more than 2500 mM, even more preferably within a range
of not less than 1500 mM and not more than 2500 mM, most preferably
within a range of not less than 2000 mM and not more than 2500
mM.
[0102] A higher concentration of the salt contained in the aqueous
solution at a high concentration can increase the specificity at
the position of the enzymatic cleavage of a chemical bond in
collagen or atelocollagen.
[0103] The salt contained in the aqueous solution at a low
concentration may have any concentration. A lower concentration is,
however, more preferable. The concentration is, for example,
preferably lower than 200 mM, more preferably not more than 150 mM,
even more preferably not more than 100 mM, even more preferably not
more than 50 mM, most preferably substantially 0 mM.
[0104] Collagen or atelocollagen may be dissolved in the aqueous
solution (for example, water) in any amount. For example, 1 part by
weight of collagen or atelocollagen is preferably dissolved in 100
parts by weight to 10000 parts by weight of the aqueous solution.
Further, 1 part by weight of collagen or atelocollagen is
preferably dissolved in 100 parts by weight to 1000 parts by weight
of the aqueous solution.
[0105] With the above feature, in a case where the enzyme has been
added to the aqueous solution, the enzyme comes into contact
efficiently with the collagen or atelocollagen. This in turn allows
the collagen or atelocollagen to be degraded efficiently with use
of the enzyme.
[0106] The enzyme may be added to the aqueous solution in any
amount. For example, 1 part by weight to 100 parts by weight of the
enzyme is preferably added to 1000 parts by weight of the collagen
or atelocollagen. With the above feature, the aqueous solution has
a high enzyme concentration. This allows the collagen or
atelocollagen to be degraded efficiently with use of the enzyme.
Further, 1 part by weight to 10 parts by weight of the enzyme is
preferably added to 100 parts by weight of the collagen or
atelocollagen.
[0107] Other conditions (for example, the pH and temperature of the
aqueous solution, and the contact period) under which the collagen
or atelocollagen is caused to be in contact with the enzyme in the
aqueous solution are not particularly limited, and may be set as
appropriate. Such other conditions are, however, preferably within
the ranges below.
[0108] (1) The aqueous solution has a pH of preferably 2.0 to 7.0,
further preferably 2.5 to 6.5. The aqueous solution can contain a
well-known buffer to have a pH kept within the above range. The
aqueous solution having a pH within the above range allows collagen
or atelocollagen to be dissolved therein uniformly, and
consequently allows an enzymatic reaction to occur efficiently.
[0109] (2) The temperature of the aqueous solution is not limited
to any particular value, and may be selected in view of the enzyme
used. The temperature is, for example, preferably within a range of
15.degree. C. to 40.degree. C., more preferably within a range of
20.degree. C. to 35.degree. C.
[0110] (3) The contact period is not limited to any particular
length, and may be selected in view of the amount of the enzyme
and/or the amount of the collagen or atelocollagen. The contact
period is, for example, preferably within a range of 1 hour to 60
days, more preferably within a range of 1 day to 7 days, further
preferably within a range of 3 days to 7 days.
[0111] A method for the present embodiment may include, as
necessary, at least one step selected from the group consisting of
a step of readjusting the pH, a step of inactivating the enzyme,
and a step of removing contaminants, after the collagen or
atelocollagen is caused to be in contact with the enzyme in the
aqueous solution.
[0112] The step of removing contaminants can be carried out by a
typical method for separating a substance. The step of removing
contaminants can be carried out by, for example, dialysis,
salting-out, gel filtration chromatography, isoelectric
precipitation, ion exchange chromatography, or hydrophobic
interaction chromatography.
[0113] The degradation step can be carried out by degrading the
collagen or atelocollagen with use of the enzyme as described
above. The collagen or atelocollagen to be degraded may be
contained in biological tissue. In other words, the degradation
step can be carried out by causing such biological tissue to be in
contact with the enzyme.
[0114] The biological tissue is not limited to any particular
tissue, and can be, for example, a dermis, a tendon, a bone, or a
fascia of a mammal or a bird, or a skin or a scale of a fish.
[0115] The biological tissue is preferably a dermis, a tendon, or a
bone from the viewpoint of maintaining high physiological activity
and the ability to produce a collagen degradation product or an
atelocollagen degradation product in a large amount.
[0116] In a case where the biological tissue is a dermis, a tendon,
or a bone, the dermis, the tendon, or the bone is preferably caused
to be in contact with the enzyme in an acidic condition. The acidic
condition is, for example, preferably a pH of 2.5 to 6.5, further
preferably a pH of 2.5 to 5.0, even further preferably a pH of 2.5
to 4.0, most preferably a pH of 2.5 to 3.5.
[0117] More specifically, in the degradation step, it is preferable
to cause a dermis, a tendon, or a bone to be in contact with the
cysteine protease so that collagen contained in the dermis, the
tendon, or the bone is caused to be in contact with the cysteine
protease. In the degradation step, it is preferable to cause the
dermis, the tendon, or the bone to be in contact with the cysteine
protease in the presence of a salt at a concentration of not less
than 200 mM. In the degradation step, it is preferable to cause the
dermis, the tendon, or the bone to be in contact with a cysteine
protease having been in contact with a salt at a concentration of
not less than 200 mM. In the degradation step, it is preferable to
cause the dermis, the tendon, or the bone to be in contact with the
cysteine protease in the presence of a salt at a concentration of
lower than 200 mM.
[0118] [2-2. Removal Step]
[0119] The removal step is a step of removing the solvent from the
collagen degradation product or the atelocollagen degradation
product obtained in the degradation step. In the removal step, not
only the solvent but also impurities such as unnecessary low
molecular weight compounds may be removed. The removal step can be
carried out, for example, by dialysis, ultrafiltration, freeze
drying, air drying, evaporator, spray drying, or a combination of
these.
[0120] The above dialysis or ultrafiltration can remove impurities
such as unnecessary low molecular weight compounds other than a
solvent (e.g., water). Dialysis or ultrafiltration may be repeated
until the amount of unnecessary low molecular weight compounds
contained in the solvent becomes negligible, and dialysis and
ultrafiltration may be carried out in combination. From the
viewpoint of preventing the collagen solid from denaturing, the
removal step is preferably carried out at a low temperature. Note
that, since the methods such as dialysis and ultrafiltration above
are well known, descriptions of such methods are omitted here.
[0121] The freeze drying, air drying, evaporator or spray drying
can remove a solvent such as water. From the viewpoint of
preventing the collagen solid from denaturing, the removal step is
preferably carried out at a low temperature. In a freezing step in
pretreatment of the freeze drying, the collagen solid may be frozen
using an ultracold freezer at -80.degree. C. Alternatively, a
program freezer may be used to cool and freeze the collagen solid
to a final temperature of -80.degree. C. Alternatively, the
collagen solid may be frozen using an ultracold freezer at
-80.degree. C. after pre-freezing the collagen solid using a
program freezer. Since the methods such as freeze drying, air
drying, evaporator, and spray drying above are well known,
descriptions of such methods are omitted here.
[0122] In the removal step (e.g., the freeze drying step or the
like), it is preferable to fill a template having an intended shape
with the collagen degradation product or the atelocollagen
degradation product and then remove the solvent from the collagen
degradation product or the atelocollagen degradation product. With
this process, the collagen solid having an intended shape can be
easily obtained. In the spray drying step, it is preferable to
spray the collagen degradation product or the atelocollagen
degradation product in the form of mist, and then remove the
solvent from the collagen degradation product or the atelocollagen
degradation product. With this process, the collagen solid in an
intended form of powder can be easily obtained.
[0123] In the removal step, it is possible that an optional
substance is added to the collagen degradation product or the
atelocollagen degradation product which has been obtained in the
degradation step to obtain a mixture, and then the solvent is
removed from the mixture. Specifically, in the removal step, it is
possible that an optional substance dissolved in a certain solvent
is added to the collagen degradation product or the atelocollagen
degradation product which has been obtained in the degradation step
to obtain a mixture, and then the solvent is removed from the
mixture. In the removal step, it is possible that a collagen solid
which has been obtained in the removal step is caused to adsorb an
optional substance. Specifically, in the removal step, it is
possible that a collagen solid which has been obtained in the
removal step is caused to absorb an optional substance dissolved in
a certain solvent. More specifically, in the removal step, it is
possible that a collagen solid which has been obtained in the
removal step is caused to absorb an optional substance dissolved in
a certain solvent, and then the solvent in which the optional
substance is dissolved is removed from the collagen solid. Further
specifically, in the removal step, it is possible that (i) a
collagen solid is obtained by removing the solvent from the
collagen degradation product or the atelocollagen degradation
product which has been obtained in the degradation step, (ii) the
collagen solid is immersed in a solvent containing an optional
substance (in other words, an optional substance dissolved in a
certain solvent), and (iii) the solvent is removed from the
collagen solid. According to the process, it is possible to produce
the collagen solid containing the optional substance.
[0124] [2-3. Other Step]
[0125] The method for producing the collagen solid in accordance
with an embodiment of the present invention can include, after the
above-described removal step, a shaping step of further applying a
shaping process (e.g., a cutting process, a polishing process, a
through hole forming process, and the like) to the collagen solid
obtained in the removal step. With this feature, the collagen solid
having an intended shape can be easily obtained. The shaping step
may be carried out according to a well-known method.
[0126] In the shaping step, an appropriate template having
projections and depressions corresponding to a shape of a shaped
product can be used. The shape of the shaped product prepared with
use of the template includes, for example, a cubic shape, a
columnar shape, a disk shape, a straight tube shape, a curved tube
shape, a screw shape, a male screw shape, a female screw shape, a
film shape, a conical shape, an arrowhead shape, a hexahedral
shape, a polyhedral shape, a polygonal column shape, a bellows
shape, and a complex shape in which two or more of these shapes are
connected to each other.
[0127] [3. Collagen Solid]
[0128] The collagen solid in accordance with an embodiment of the
present invention can be prepared by the production method
described in the section of [2. Method for producing collagen
solid] above. The collagen solid in accordance with an embodiment
of the present invention includes a collagen-cysteine protease
degradation product or an atelocollagen-cysteine protease
degradation product. The following description will discuss the
individual features. Note that, in regard to the features described
above in the section of [2. Method for producing collagen solid],
descriptions of such features will be omitted below.
[0129] [3-1. Properties of Collagen Solid]
[0130] A density of the collagen solid in accordance with an
embodiment of the present invention is preferably approximately 50
mg/cm.sup.3 or more, more preferably approximately 50 mg/cm.sup.3
to approximately 400 mg/cm.sup.3, more preferably approximately 50
mg/cm.sup.3 to approximately 350 mg/cm.sup.3, more preferably
approximately 80 mg/cm.sup.3 to approximately 350 mg/cm.sup.3, more
preferably approximately 80 mg/cm.sup.3 to approximately 300
mg/cm.sup.3, more preferably approximately 100 mg/cm.sup.3 to
approximately 300 mg/cm.sup.3, more preferably approximately 120
mg/cm.sup.3 to approximately 300 mg/cm.sup.3, more preferably
approximately 120 mg/cm.sup.3 to approximately 280 mg/cm.sup.3,
more preferably approximately 140 mg/cm.sup.3 to approximately 280
mg/cm.sup.3, more preferably approximately 140 mg/cm.sup.3 to
approximately 260 mg/cm.sup.3, more preferably approximately 140
mg/cm.sup.3 to approximately 240 mg/cm.sup.3, most preferably
approximately 140 mg/cm.sup.3 to approximately 220 mg/cm.sup.3.
[0131] A method of measuring the density of the collagen solid is
not particularly limited, and for example, the density can be
measured by a method described in Examples described later.
[0132] A tangent modulus of the collagen solid in accordance with
an embodiment of the present invention is preferably approximately
90 kPa or more, more preferably approximately 90 kPa to
approximately 40000 kPa, more preferably approximately 90 kPa to
approximately 35000 kPa, more preferably approximately 150 kPa to
approximately 35000 kPa, more preferably approximately 200 kPa to
approximately 35000 kPa, more preferably approximately 200 kPa to
approximately 30000 kPa, more preferably approximately 200 kPa to
approximately 25000 kPa, more preferably approximately 250 kPa to
approximately 25000 kPa, more preferably approximately 300 kPa to
approximately 25000 kPa, more preferably approximately 300 kPa to
approximately 20000 kPa, more preferably approximately 300 kPa to
approximately 15000 kPa, most preferably approximately 300 kPa to
approximately 10000 kPa.
[0133] A method of measuring the tangent modulus of the collagen
solid is not particularly limited, and for example, the tangent
modulus can be measured by a method described in Examples described
later.
[0134] The collagen solid in accordance with an embodiment of the
present invention can have the density described above, can have
the tangent modulus described above, and can have both the density
and the tangent modulus described above.
[0135] An amount of each of the collagen-cysteine protease
degradation product and the atelocollagen-cysteine protease
degradation product contained in the collagen solid in accordance
with an embodiment of the present invention is not particularly
limited. However, a larger amount of these degradation products is
preferable because the strength of the collagen solid is improved.
For example, a total amount of each of the degradation products in
the collagen solid in accordance with an embodiment of the present
invention can be preferably 0.1% by weight to 100% by weight, more
preferably 50% by weight to 100% by weight, more preferably 90% by
weight to 100% by weight, most preferably 100% by weight.
[0136] To the collagen solid in accordance with an embodiment of
the present invention, components other than the collagen-cysteine
protease degradation product and the atelocollagen-cysteine
protease degradation product can be added. These components are not
particularly limited. Examples of these components include elements
(e.g., calcium, magnesium, potassium, sodium, chloride, zinc, iron,
and copper, or ions thereof), inorganic acids (phosphoric acid,
acetic acid, and carbonic acid, or ions thereof), organic acids
(pyruvic acid, acetyl-CoA, citric acid, oxalacetic acid, succinic
acid, and fumaric acid, or ions thereof), low molecular weight
compounds (e.g., CaCO.sub.3) nucleic acids (DNA, RNA, plasmids),
nucleosides, nucleotides, ATP, GTP, NADH, FADH.sub.2, siRNA, miRNA,
lipids, amino acids, proteins, cytokines, growth factors (e.g.,
FGF, bFGF, VEGF, BMP, TGF-.beta., PDGF, HGF, and IGF),
monosaccharides (glucose, fucose, glucosamine), polysaccharides
(hyaluronic acid, trehalose, amylose, pectin, cellulose, glycogen,
starch, and chitin), chemically synthesized drugs, natural drugs,
enzymes, hormones (testosterone, dihydrotestosterone, estrone,
estradiol, progesterone, luteinizing hormone, follicle-stimulating
hormone, thyroid hormone), antibiotics, anticancer drugs,
proteoglycans, antibodies, exosomes, and cytoclastic components,
mixtures thereof, and the like.
[0137] In a case where the collagen solid in accordance with an
embodiment of the present invention includes components other than
the collagen-cysteine protease degradation product and the
atelocollagen-cysteine protease degradation product, the collagen
solid can be obtained as follows: (i) the collagen-cysteine
protease degradation product and/or the atelocollagen-cysteine
protease degradation product, a solvent, and components other than
the collagen-cysteine protease degradation product and the
atelocollagen-cysteine protease degradation product are mixed and
then the solvent is evaporated to obtain a collagen solid which
contains other components or (ii) the collagen-cysteine protease
degradation product and/or the atelocollagen-cysteine protease
degradation product and a solvent are mixed and dried to obtain a
collagen solid, then the collagen solid thus obtained is caused to
absorb components other than the collagen-cysteine protease
degradation product and the atelocollagen-cysteine protease
degradation product, and then unnecessary solvent and the like are
evaporated to obtain a collagen solid containing other components.
Alternatively, the collagen-cysteine protease degradation product
or the atelocollagen-cysteine protease degradation product is mixed
with a plurality of components other than the collagen-cysteine
protease degradation product or the atelocollagen-cysteine protease
degradation product, and then unnecessary solvent and the like are
evaporated to obtain a collagen solid containing the plurality of
components.
[0138] In the collagen solid in accordance with an embodiment of
the present invention, components other than the collagen-cysteine
protease degradation product and the atelocollagen-cysteine
protease degradation product can be contained in a total amount of
0% by weight to 99.9% by weight, 0% by weight to 50% by weight, 0%
by weight to 10% by weight, or 0% by weight.
[0139] The collagen solid in accordance with an embodiment of the
present invention can have been processed to have an intended
shape. Examples of the shape include a disk shape, a tube shape, a
columnar shape, a conical shape, an arrowhead shape, a hexahedral
shape, a polyhedral shape, a polygonal column shape, a bellows
shape, a screw shape, a male screw shape, a female screw shape and
a complex shape in which two or more of these shapes are connected
to each other. Of course, however, the present invention is not
limited to these shapes.
[0140] [2-2. Collagen-Cysteine Protease Degradation Product and
Atelocollagen-Cysteine Protease Degradation Product Contained in
Collagen Solid]
[0141] Each of the collagen-cysteine protease degradation product
and the atelocollagen-cysteine protease degradation product can
contain at least a part of a triple helical domain of collagen. The
degradation product may, in other words, contain the entire triple
helical domain of collagen or a portion of the triple helical
domain.
[0142] More specifically, each of the collagen-cysteine protease
degradation product and the atelocollagen-cysteine protease
degradation product can be a degradation product of collagen or
atelocollagen which degradation product results from:
[0143] cleavage of a chemical bond between X.sub.1 and X.sub.2,
between X.sub.2 and G, between G and X.sub.3, between X.sub.4 and
G, or between X.sub.6 and G in an amino acid sequence in (1) below
within the triple helical domain of collagen or atelocollagen;
[0144] cleavage of a chemical bond between X.sub.1 and X.sub.2,
between X.sub.2 and G, between G and X.sub.3, between X.sub.4 and
G, between X.sub.6 and G, between G and X.sub.7, or between
X.sub.14 and G in an amino acid sequence in (2) below within the
triple helical domain of collagen or atelocollagen; or
[0145] cleavage of a chemical bond between Y.sub.1 and Y.sub.2 in
an amino acid sequence in (3) below at an amino terminus of the
triple helical domain of collagen or atelocollagen.
TABLE-US-00001 (SEQ ID NO: 1) (1)
-G-X.sub.1-X.sub.2-G-X.sub.3-X.sub.4-G-X.sub.5-X.sub.6-G-, (SEQ ID
NO: 2) (2)
-G-X.sub.1-X.sub.2-G-X.sub.3-X.sub.4-G-X.sub.5-X.sub.6-G-X.sub.7-X.su-
b.8-G-X.sub.9-X.sub.10-G- X.sub.11-X.sub.12-G-X.sub.13-X.sub.14-G-,
(SEQ ID NO: 3) (3)
-Y.sub.1-Y.sub.2-Y.sub.3-G-Y.sub.4-Y.sub.5-G-Y.sub.6-Y.sub.7-G-Y.sub.-
8-Y.sub.9-G-,
[0146] where G represents glycine, and X.sub.1 to X.sub.14 and
Y.sub.1 to Y.sub.9 each represent any amino acid.
[0147] The term "triple helical domain" as used in the present
specification intends to mean a domain that (i) contains not fewer
than 3, preferably not fewer than 80, more preferably not fewer
than 100, more preferably not fewer than 200, more preferably not
fewer than 300, units of amino acid sequences in tandem each of
which units is represented as "Gly-X-Y" (where X and Y each
represent an amino acid) and that (ii) contributes to formation of
a helical structure.
[0148] The cleavage of a chemical bond within the triple helical
domain may occur in any of a plurality of kinds of polypeptide
chains included in the collagen. The cleavage of a chemical bond
may occur in, for example, any of the following polypeptide chains:
the .alpha.1 chain, the .alpha.2 chain, and the .alpha.3 chain. The
cleavage of a chemical bond occurs preferably in at least one of
the .alpha.1 chain and the .alpha.2 chain among the above
polypeptide chains. The cleavage of a chemical bond occurs further
preferably in the .alpha.1 chain among the above polypeptide
chains.
[0149] Each of the collagen-cysteine protease degradation product
and the atelocollagen-cysteine protease degradation product may
contain three polypeptide chains in a helical structure. Each of
the collagen-cysteine protease degradation product and the
atelocollagen-cysteine protease degradation product may
alternatively contain three polypeptide chains that are not in a
helical structure entirely or partially. Whether the three
polypeptide chains are in a helical structure can be determined by
a publicly known method (for example, by observing a circular
dichroism spectrum of the degradation product).
[0150] Each of the collagen-cysteine protease degradation product
and the atelocollagen-cysteine protease degradation product
basically contains three polypeptide chains. The cleavage of a
chemical bond may occur in one, two, or all of the three
polypeptide chains.
[0151] In a case where the degradation product of collagen or
atelocollagen contains three polypeptide chains in a helical
structure, a plurality of helical structures may form a meshwork
assembly or filamentous assembly. The term "meshwork" as used in
the present specification intends to describe a structure of
molecules connected to one another through, for example, hydrogen
bonding, electrostatic interaction, or van der Waals bonding to
form a three-dimensional mesh and openings therein. The term
"filamentous" as used in the present specification intends to
describe a substantially linear structure of molecules connected to
one another through, for example, hydrogen bonding, electrostatic
interaction, or van der Waals bonding. The term "assembly" as used
in the present specification intends to mean a structural unit of
two or more molecules of an identical kind that bond to one another
not through covalent bonding but through interaction with one
another. Whether a meshwork or filamentous assembly is present can
be determined by observing the degradation product with an electron
microscope.
[0152] The amino acid sequence in (1) or (2) above may be at any
position within the triple helical domain. The amino acid sequence
in (1) or (2) above may be, for example, at a position away from
the two terminuses of the triple helical domain, but is preferably
at the amino terminus of the triple helical domain. Stated
differently, that "G" in the amino acid sequence in (1) or (2)
above which is closest to the amino terminus preferably corresponds
to that "G" within the triple helical domain which is closest to
the amino terminus.
[0153] Each of the amino acid sequences in (1), (2) and (3) may be
connected, at the amino terminus of each of the amino acid
sequences in (1), (2) and (3), to not fewer than 1, not fewer than
5, not fewer than 10, not fewer than 50, not fewer than 100, not
fewer than 150, not fewer than 200, not fewer than 250, or not
fewer than 300, units of amino acid sequences in tandem each of
which units is represented as "Gly-X-Y" (where X and Y each
represent an amino acid). Each of the amino acid sequences in (1),
(2) and (3) may be connected, at the carboxyl terminus of each of
the amino acid sequences in (1), (2) and (3), to not fewer than 1,
not fewer than 5, not fewer than 10, not fewer than 50, not fewer
than 100, not fewer than 150, not fewer than 200, not fewer than
250, or not fewer than 300, units of amino acid sequences in tandem
each of which units is represented as "Gly-X-Y" (where X and Y each
represent an amino acid).
[0154] X.sub.1 to X.sub.6 can each be any amino acid, and are each
not limited to any particular kind. At least two of X.sub.1 to
X.sub.6 may be amino acids of an identical kind. X.sub.1 to X.sub.6
may alternatively be amino acids all of which differ from one
another in kind.
[0155] X.sub.1 to X.sub.6 may each be, for example, any of the
following amino acids: glycine, alanine, valine, leucine,
isoleucine, serine, threonine, tyrosine, cysteine, methionine,
aspartic acid, asparagine, glutamic acid, glutamine, arginine,
lysine, histidine, phenylalanine, tyrosine, tryptophan,
hydroxyproline, and hydroxylysine.
[0156] Further specifically, X.sub.1 to X.sub.6 may be such that
X.sub.1, X.sub.3, and X.sub.5 are identical amino acids, while the
others are different amino acids.
[0157] Further specifically, X.sub.1 to X.sub.6 may be such that at
least one selected from the group consisting of X.sub.1, X.sub.3,
and X.sub.5 is proline, while the others are each any amino
acid.
[0158] Further specifically, X.sub.1 to X.sub.6 may be such that
X.sub.1 is proline, while X.sub.2 to X.sub.6 are each any amino
acid.
[0159] Further specifically, X.sub.1 to X.sub.6 may be such that
X.sub.1 and X.sub.3 are each proline, while X.sub.2 and X.sub.4 to
X.sub.6 are each any amino acid.
[0160] Further specifically, X.sub.1 to X.sub.6 may be such that
X.sub.1, X.sub.3, and X.sub.5 are each proline, while X.sub.2,
X.sub.4, and X.sub.6 are each any amino acid.
[0161] Further specifically, X.sub.1 to X.sub.6 may be such that
(i) X.sub.1, X.sub.3, and X.sub.5 are each proline, (ii) X.sub.2 is
an amino acid containing a sulfur atom in a side chain (for
example, cysteine or methionine) or an amino acid containing a
hydroxyl group in a side chain (for example, hydroxyproline,
hydroxylysine, or serine), and (iii) X.sub.4 and X.sub.6 are each
any amino acid.
[0162] Further specifically, X.sub.1 to X.sub.6 may be such that
(i) X.sub.1, X.sub.3, and X.sub.5 are each proline, (ii) X.sub.2 is
an amino acid containing a sulfur atom in a side chain (for
example, cysteine or methionine), (iii) X.sub.4 is an amino acid
having an aliphatic side chain (for example, glycine, alanine,
valine, leucine, or isoleucine) or an amino acid containing a
hydroxyl group in a side chain (for example, hydroxyproline,
hydroxylysine, or serine), and (iv) X.sub.6 is any amino acid.
[0163] Further specifically, X.sub.1 to X.sub.6 may be such that
X.sub.1, X.sub.3, and X.sub.5 are each proline, (ii) X.sub.2 is an
amino acid containing a sulfur atom in a side chain (for example,
cysteine or methionine), (iii) X.sub.4 is an amino acid having an
aliphatic side chain (for example, glycine, alanine, valine,
leucine, or isoleucine) or an amino acid containing a hydroxyl
group in a side chain (for example, hydroxyproline, hydroxylysine,
or serine), and (iv) X.sub.6 is an amino acid containing a base in
a side chain (for example, arginine, lysine, or histidine).
[0164] Further specifically, X.sub.1 to X.sub.6 may be such that
(i) X.sub.1, X.sub.3, and X.sub.5 are each proline, (ii) X.sub.2 is
methionine, (iii) X.sub.4 is alanine or serine, and (iv) X.sub.6 is
arginine.
[0165] In the amino acid sequence in (2) above, X.sub.1 to X.sub.6
may be identical in arrangement to the above X.sub.1 to X.sub.6,
respectively. The following description will discuss detailed
arrangements of X.sub.7 to X.sub.14.
[0166] X.sub.7 to X.sub.14 can each be any amino acid, and are each
not limited to any particular kind. At least two of X.sub.7 to
X.sub.14 may be amino acids of an identical kind. X.sub.7 to
X.sub.14 may alternatively be amino acids all of which differ from
one another in kind.
[0167] X.sub.7 to X.sub.14 may each be, for example, any of the
following amino acids: glycine, alanine, valine, leucine,
isoleucine, serine, threonine, tyrosine, cysteine, methionine,
aspartic acid, asparagine, glutamic acid, glutamine, arginine,
lysine, histidine, phenylalanine, tyrosine, tryptophan,
hydroxyproline, and hydroxylysine.
[0168] Further specifically, X.sub.7 to X.sub.14 may be such that
X.sub.6, X.sub.9. X.sub.10, X.sub.12, and X.sub.13 are identical
amino acids, while the others are different amino acids.
[0169] Further specifically, X.sub.7 to X.sub.14 may be such that
at least one selected from the group consisting of X.sub.6,
X.sub.9, X.sub.10, X.sub.12, and X.sub.13 is proline or
hydroxyproline, while the others are each any amino acid.
[0170] Further specifically, X.sub.7 to X.sub.14 may be such that
X.sub.8 is proline or hydroxyproline, while the others are each any
amino acid.
[0171] Further specifically, X.sub.7 to X.sub.14 may be such that
X.sub.6 and X.sub.9 are each proline or hydroxyproline, while the
others are each any amino acid.
[0172] Further specifically, X.sub.7 to X.sub.14 may be such that
X.sub.8, X.sub.9, and X.sub.10 are each proline or hydroxyproline,
while the others are each any amino acid.
[0173] Further specifically, X.sub.7 to X.sub.14 may be such that
X.sub.8, X.sub.9, X.sub.10, and X.sub.12 are each proline or
hydroxyproline, while the others are each any amino acid.
[0174] Further specifically, X.sub.7 to X.sub.14 may be such that
X.sub.8, X.sub.9, X.sub.10, X.sub.12, and X.sub.13 are each proline
or hydroxyproline, while the others are each any amino acid.
[0175] Further specifically, X.sub.7 to X.sub.14 may be such that
(i) X.sub.6, X.sub.9, X.sub.10, X.sub.12, and X.sub.13 are each
proline or hydroxyproline, (ii) X.sub.7 is an amino acid having an
aliphatic side chain (for example, glycine, alanine, valine,
leucine, or isoleucine), and (iii) the others are each any amino
acid.
[0176] Further specifically, X.sub.7 to X.sub.14 may be such that
(i) X.sub.6, X.sub.9, X.sub.10, X.sub.12, and X.sub.13 are each
proline or hydroxyproline, (ii) X.sub.7 and X.sub.11 are each an
amino acid having an aliphatic side chain (for example, glycine,
alanine, valine, leucine, or isoleucine), and (iii) the rest is any
amino acid.
[0177] Further specifically, X.sub.7 to X.sub.14 may be such that
(i) X.sub.6, X.sub.9, X.sub.10, X.sub.12, and X.sub.13 are each
proline or hydroxyproline, (ii) X.sub.7 and X.sub.11 are each an
amino acid having an aliphatic side chain (for example, glycine,
alanine, valine, leucine, or isoleucine), and (iii) X.sub.14 is an
amino acid having a hydrophilic and non-dissociative side chain
(serine, threonine, asparagine, or glutamine).
[0178] Further specifically, X.sub.7 to X.sub.14 may be such that
(i) X.sub.6, X.sub.9, X.sub.10, X.sub.12, and X.sub.13 are each
proline or hydroxyproline, (ii) X.sub.7 is leucine, (iii) X.sub.11
is alanine, and (iv) X.sub.14 is glutamine.
[0179] The amino acid sequence in (3) above is positioned at the
amino terminus of the triple helical domain. This means that (i) G
between Y.sub.3 and Y.sub.4 indicates glycine which is within the
triple helical domain and is closest to the amino terminus and that
(ii) Y.sub.1, Y.sub.2, and Y.sub.3 indicate amino acids which are
in a plurality of kinds of polypeptide chains included in the
collagen and are positioned closer to the amino terminus than the
triple helical domain.
[0180] Y.sub.1 to Y.sub.9 can each be any amino acid, and are each
not limited to any particular kind. At least two of Y.sub.1 to
Y.sub.9 may be amino acids of an identical kind. Y.sub.1 to Y.sub.9
may alternatively be amino acids all of which differ from one
another in kind.
[0181] Y.sub.1 to Y.sub.9 may each be, for example, any of the
following amino acids: glycine, alanine, valine, leucine,
isoleucine, serine, threonine, tyrosine, cysteine, methionine,
aspartic acid, asparagine, glutamic acid, glutamine, arginine,
lysine, histidine, phenylalanine, tyrosine, tryptophan,
hydroxyproline, and hydroxylysine.
[0182] Further specifically, Y.sub.1 to Y.sub.3 may be such that
Y.sub.3 is proline, while Y.sub.1 and Y.sub.2 are each any amino
acid.
[0183] Further specifically, Y.sub.1 to Y.sub.3 may be such that
Y.sub.3 is proline, while Y.sub.1 and Y.sub.2 are each an amino
acid having an aliphatic side chain (for example, glycine, alanine,
valine, leucine, or isoleucine) or an amino acid containing a
hydroxyl group in a side chain (hydroxyproline, hydroxylysine, or
serine).
[0184] Further specifically, Y.sub.1 to Y.sub.3 may be such that
(i) Y.sub.3 is proline, (ii) Y.sub.1 is alanine or serine, and
(iii) Y.sub.2 is valine.
[0185] In any of the above cases, Y.sub.4 to Y.sub.9 are not
particularly limited in terms of specific arrangements. Y.sub.4 to
Y.sub.9 may be such that (i) Y.sub.4 and X.sub.1 are identical
amino acids, (ii) Y.sub.5 and X.sub.2 are identical amino acids,
(iii) Y.sub.9 and X.sub.3 are identical amino acids, (iv) Y.sub.7
and X.sub.4 are identical amino acids, (v) Y.sub.6 and X.sub.5 are
identical amino acids, and (vi) Y.sub.9 and X.sub.6 are identical
amino acids.
[0186] More specifically, X.sub.1 and Y.sub.4 can each be proline,
X.sub.2 and Y.sub.5 can each be methionine, X.sub.3 and Y.sub.6 can
each be proline or leucine, X.sub.4 and Y.sub.7 can each be
alanine, serine, or methonine, X.sub.5 and Y.sub.8 can each be
proline or serine, X.sub.6 and Y.sub.9 can each be arginine,
X.sub.7 to X.sub.14 and Y.sub.1 to Y.sub.3 can each be any amino
acid.
[0187] [4. Biomaterial and Ex Vivo Material]
[0188] Each of the biomaterial and the ex vivo material in
accordance with an embodiment of the present invention contains the
above described collagen solid.
[0189] The biomaterial can be implanted in a biological tissue
(e.g., bone). The biomaterial in accordance with an embodiment of
the present invention can be a biomaterial for biological tissue
regeneration, or can be a biomaterial for biological tissue
repairing. More specifically, the biomaterial in accordance with an
embodiment of the present invention can be a biomaterial for bone
regeneration (in other words, a bone regeneration material)
containing the collagen solid described above. More specifically,
the biomaterial in accordance with an embodiment of the present
invention can be a biomaterial which contains the collagen solid
described above and is used in bone regeneration for treating bone
injury or a biomaterial which contains the collagen solid described
above and is used in bone regeneration for treating bone defect. As
also shown in Examples described later, the biomaterial in
accordance with an embodiment of the present invention can
effectively regenerate a bone.
[0190] The ex vivo material is not particularly limited, provided
that the ex vivo material is used outside biological tissues. The
ex vivo material in accordance with an embodiment of the present
invention can be a substrate for cell culture or a cell culture
substrate composed of a highly dense collagen solid whose shape can
be processed. The ex vivo material in accordance with an embodiment
of the present invention can be a cell culture substrate containing
the highly dense collagen solid described above. More specifically,
the ex vivo material in accordance with an embodiment of the
present invention can be a film-like or membrane-like cell culture
substrate or a cubic cell culture substrate containing the highly
dense collagen solid described above. The ex vivo material in
accordance with an embodiment of the present invention makes it
possible to effectively culture cells on the ex vivo material.
[0191] To the biomaterial and the ex vivo material in accordance
with an embodiment of the present invention, components other than
the collagen solid can be added. These components are not
particularly limited. Examples of these components include elements
(e.g., calcium, magnesium, potassium, sodium, chloride, zinc, iron,
and copper, or ions thereof), inorganic acids (phosphoric acid,
acetic acid, and carbonic acid, or ions thereof), organic acids
(pyruvic acid, acetyl-CoA, citric acid, oxalacetic acid, succinic
acid, and fumaric acid, or ions thereof), low molecular weight
compounds (e.g., CaCO.sub.3), nucleic acids (DNA, RNA, plasmids),
nucleosides, nucleotides, ATP, GTP, NADH, FADH.sub.2, siRNA, miRNA,
lipids, amino acids, proteins, cytokines, growth factors (e.g.,
FGF, bFGF, VEGF, BMP, TGF-.beta., PDGF, HGF, and IGF),
monosaccharides (glucose, fucose, glucosamine), polysaccharides
(hyaluronic acid, trehalose, amylose, pectin, cellulose, glycogen,
starch, and chitin), chemically synthesized drugs, natural drugs,
enzymes, hormones (testosterone, dihydrotestosterone, estrone,
estradiol, progesterone, luteinizing hormone, follicle-stimulating
hormone, thyroid hormone), antibiotics, anticancer drugs,
proteoglycans, antibodies, exosomes, and cytoclastic components,
mixtures thereof, and the like.
[0192] Each of the biomaterial and the ex vivo material in
accordance with an embodiment of the present invention can contain
the collagen solid in an amount of preferably 0.1% by weight to
100% by weight, more preferably 50% by weight to 100% by weight,
more preferably 90% by weight to 100% by weight, more preferably
95% by weight to 100% by weight, most preferably 100% by weight. In
the biomaterial in accordance with an embodiment of the present
invention, components other than the collagen solid can be
contained in a total amount of 0% by weight to 99.9% by weight, 0%
by weight to 50% by weight, 0% by weight to 10% by weight, 0% by
weight to 5% by weight, or 0% by weight.
[0193] A method of using the biomaterial obtained as described
above can include, for example, (i) a cleaning step of cleaning and
sterilizing a biomaterial containing a collagen solid, (ii) an
implantation step of implanting the cleaned biomaterial into a
biological tissue of interest, and (iii) an evaluation step of
evaluating a degree of progression of bone adhesion at a site where
the biomaterial has been implanted in the biological tissue.
[0194] Examples of the cleaning step include cleaning of the
biomaterial with an organic solvent (70% ethanol, acetone, or the
like), cleaning of the biomaterial with sterile water, and
sterilization of the biomaterial by UV-irradiation. Examples of the
implantation step include a process of implanting the biomaterial
into a biological tissue of interest, and a process of filling a
biological tissue of interest with the biomaterial. Examples of the
evaluation step include evaluation with medical imaging technology,
mechanical evaluation, immunohistochemical evaluation (i.e.,
evaluation by immunostaining) and histological evaluation.
[0195] In the evaluation with medical imaging technology, for
example, images of the site at which the biomaterial has been
implanted are obtained by radiography or computed tomography, the
images are classified in accordance with predetermined evaluation
criteria, and a degree of repair is evaluated based on the
classification.
[0196] In the mechanical evaluation, for example, mechanical
strength of the site at which the biomaterial has been implanted is
obtained by a three-point bending extrusion tester, the images are
classified in accordance with predetermined evaluation criteria,
and a degree of repair is evaluated based on the
classification.
[0197] In the immunohistochemical evaluation (i.e., evaluation by
immunostaining), a target antigen is detected with use of a
specific antibody in order to visualize the presence and
localization of a component of interest on the tissue with a
microscope. For example, an amount of presence at the site at which
the biomaterial has been implanted is obtained with an
anti-osteocalcin antibody, the images are classified in accordance
with predetermined evaluation criteria, and a degree of repair is
evaluated based on the classification. In regard to the antibody, a
plurality of antibodies can be used together or individually in
accordance with a degree of maturity of a bone regeneration tissue.
Note, however, that the present invention is not limited to
this.
[0198] In the histological evaluation, images of the site at which
the biomaterial has been implanted are obtained after HE stain or
SO stain, the images are classified in accordance with
predetermined evaluation criteria, and a degree of repair is
evaluated based on the classification.
[0199] A method of using the ex vivo material obtained as described
above can include, for example, (i) a cleaning step of cleaning and
sterilizing an ex vivo material containing the collagen solid: (ii)
a shaping step of forming the cleaned ex vivo material into a shape
for intended cell culture; (iii) a culture step of seeding and
culturing cells on the shaped ex vivo material; and (iv) an
evaluation step of evaluating morphology and function of cells.
[0200] Examples of the cleaning step include cleaning of the ex
vivo material with an organic solvent (70% ethanol, acetone, and
the like), cleaning of the ex vivo material with sterile water, and
sterilization of the ex vivo material by UV-irradiation. Examples
of the shaping step include a process of injecting the ex vivo
material into an appropriate template to obtain an intended shaped
product, a process of filling the template with the ex vivo
material, and a process of freeze-drying the ex vivo material.
Examples of the evaluation step include image evaluation of cell
morphology, moving speed evaluation, immunohistochemical evaluation
(i.e., evaluation by immunostaining), evaluation of protein
expression level, and evaluation of gene expression level. In the
culture step, a culture medium, a culture temperature, and the like
can be set in accordance with cells to be cultured, and the culture
step can be carried out according to a known method.
EXAMPLES
Example 1. Preparation of the Columnar Collagen Solid
[0201] Using actinidain, which is a cysteine protease, a
degradation process was carried out on type I collagen derived from
a pig at 20.degree. C. for 7 days. Thus, a collagen-cysteine
protease degradation product "LASCol" (low adhesive scaffold
collagen (type 1)) in accordance with an aspect of the present
invention having a triple-stranded structure was obtained. The
LASCol was dialyzed with respect to 10 million-fold ultrapure water
to remove impurities and the like, and then the solution after
dialysis was placed in an appropriate container and frozen in an
ultracold freezer at -80.degree. C. After that, the LASCol was
freeze-dried in a freeze dryer (FDU-2200 available from Tokyo
Rikakikai Co, Ltd.)
[0202] Ultrapure water was then added to 100 mg of the freeze-dried
LASCol such that the LASCol is contained at predetermined
concentrations (i.e., 10 mg/mL, 30 mg/mL, 50 mg/mL, 100 mg/mL, 150
mg/mL, 180 mg/mL), and solutions thus obtained were left to stand
for 3 days to 10 days in a refrigerator at 0.degree. C. to
10.degree. C. During the above periods, the solutions were gently
mixed without being bubbled, and the freeze-dried LASCol was thus
completely dissolved.
[0203] Each of the solutions in which the LASCol was completely
dissolved was put into a columnar template (diameter: 2.5 mm to 3.0
mm, length: 4 mm to 7 mm) and frozen at -80.degree. C. The frozen
product was placed in a chamber of the freeze dryer (FDU-2200
available from Tokyo Rikakikai Co, Ltd.) to completely remove water
from the completely frozen solution by sublimation under conditions
of approximately -85.degree. C. and 2.0 Pa to 2.5 Pa. Then, a
LASCol solid obtained after freeze drying was taken out from the
columnar template to obtain a columnar LASCol solid.
[0204] As a comparative example, commercially available
pepsin-treated type I collagen (Cellmatrix Type I-C, available from
Nitta Gelatin Inc.) (in other words, "atelocollagen" in which
collagen was degraded with pepsin) was used, and a columnar
atelocollagen solid was prepared in a manner similar to that
described above. Note that ultrapure water was added to
atelocollagen so as to obtain a solution having a high
atelocollagen concentration. However, only a solution having an
atelocollagen concentration of 18 mg/mL or 20 mg/mL was obtained.
In other words, it was not physically possible to obtain a solution
having an atelocollagen concentration of more than 20 mg/mL in a
state in which atelocollagen was completely dissolved.
Example 2. Scanning Electron Microscopy (SEM) Observation
[0205] A razor was used to cut the columnar LASCol solid or the
columnar atelocollagen solid in half in a longitudinal direction.
Then, a piece of the columnar LASCol solid or the columnar
atelocollagen solid was stuck on a sample table for SEM with a
carbon double-faced tape for SEM so that a cut surface faced
upward. Next, platinum-palladium was vapor-deposited to have a
thickness of 5 nm on the cut surface with a vapor deposition device
(Ion Sputter E-1030, available from Hitachi High-Technologies
Corporation) to obtain an observation sample.
[0206] The observation sample was imaged using a scanning electron
microscope (SU3500, Hitachi High-Technologies Corporation)
(acceleration voltage: 5 kV, spot intensity: 30). The results are
shown in FIGS. 1 through 3.
[0207] FIGS. 1 and 2 show cross-sectional images of LASCol, where
collagen densities are (a) 10 mg/cm.sup.3, (b) 53 mg/cm.sup.3, (c)
81 mg/cm.sup.3, (d) 150 mg/cm.sup.3, (e) 209 mg/cm.sup.3, and (f)
264 mg/cm.sup.3.
[0208] FIG. 3 shows cross-sectional images of the atelocollagen
solid of Comparative Example, where collagen densities are (a) 18
mg/cm.sup.3 and (b) 26 mg/cm.sup.3.
[0209] From FIGS. 1 through 3, it can be seen that the LASCol
solids are more densely packed with the degradation product, as
compared with the atelocollagen solids. From FIGS. 1 and 2, it was
observed that, when the concentration of collagen dissolved in the
solvent increases, the inside of the LASCol solid became
denser.
Example 3. Preparation of Bellows-Shaped LASCol Solid
[0210] In a manner similar to that of Example 1 above, ultrapure
water was added to freeze-dried LASCol so that a concentration of
the LASCol became 100 mg/mL, and the LASCol was completely
dissolved. A solution in which the LASCol was completely dissolved
was put into a bellows-shaped bent tubular template 1 (root
diameter (D1): 4.5 mm, outer diameter (D2): 5.1 mm, length (L1):
20.1 mm, (L2): 16.5 mm) or a bellows-shaped stretched tubular
template 2 (root diameter (D1): 4.5 mm, outer diameter (D2): 5.1
mm, length (L3): 27.5 mm) and was frozen at -80.degree. C.
[0211] With use of the freeze dryer (FDU-2200 available from Tokyo
Rikakikai Co, Ltd.), water was completely removed from the
completely frozen solution by sublimation under conditions of
approximately -85.degree. C. and 2.0 Pa to 2.5 Pa. Then, a LASCol
solid obtained after freeze drying was taken out from the tubular
template 1 or the tubular template 2 to obtain a bellows-shaped
LASCol solid. FIG. 4 shows images of the bellows-shaped LASCol
solids.
[0212] (a) of FIG. 4 shows a bent bellows-shaped LASCol solid. (b)
of FIG. 4 shows a stretched bellows-shaped LASCol solid. It can be
seen that the LASCol in accordance with an embodiment of the
present invention can be formed into intended shapes.
Example 4. Preparation of Small-Diameter-Tubular LASCol Solid
[0213] In a manner similar to that of Example 1 above, freeze-dried
LASCol was completely dissolved in ultrapure water so that a
concentration of the LASCol became 100 mg/mL or 150 mg/mL. A
solution in which the LASCol was completely dissolved was put into
a tubular template 3 (inner diameter (D1): 4.0 mm, outer diameter
(D2): 6.7 mm, length (L4): 7.5 mm), a tubular template 4 (inner
diameter (D1): 2.2 mm, outer diameter (D2): 3.4 mm, length (L5):
29.1 mm), or a tubular template 5 (inner diameter (D1): 0.95 mm,
outer diameter (D2): 2.50 mm, length (L6): 24.5 mm), and was frozen
at -80.degree. C.
[0214] With use of the freeze dryer (FDU-2200 available from Tokyo
Rikakikai Co, Ltd.), water was completely removed from the
completely frozen solution by sublimation under conditions of
approximately -85.degree. C. and 2.0 Pa to 2.5 Pa. Then, a LASCol
solid obtained after freeze drying was taken out from the tubular
template 3, the tubular template 4, or the tubular template 5 to
obtain a small-diameter-tubular LASCol solid. FIG. 5 shows images
of the small-diameter-tubular LASCol solids.
[0215] (a) of FIG. 5 shows a small-diameter-tubular LASCol solid
obtained by filling the tubular template 3 with a solution
containing LASCol at a collagen concentration of 100 mg/mL. (b) of
FIG. 5 shows a small-diameter-tubular LASCol solid obtained by
filling the tubular template 4 with a solution containing LASCol at
a collagen concentration of 150 mg/mL. (c) of FIG. 5 shows a
small-diameter-tubular LASCol solid obtained by filling the tubular
template 5 with a solution containing LASCol at a collagen
concentration of 150 mg/mL.
[0216] From (a) through (c) of FIG. 5, it can be seen that, at any
collagen concentration in LASCol, small-diameter-tubular LASCol
solids formed into a very small hollow tubular shape are
obtained.
Example 5. Strength Test of Columnar LASCol Solid
[0217] In a manner similar to that of Example 1, a columnar LASCol
solid (diameter: 2.5 mm) was obtained by freeze-drying a solution
containing LASCol having a predetermined collagen concentration (30
mg/mL, 50 mg/mL, 100 mg/mL, 150 mg/mL, 180 mg/mL). Similarly, a
columnar atelocollagen solid (diameter: 2.5 mm) was obtained by
freeze-drying a solution containing atelocollagen at a
predetermined collagen concentration (20 mg/mL). The columnar
LASCol solids and the columnar atelocollagen solid were cut by a
razor to obtain columnar pieces having a diameter of 5 mm and a
length of 5 mm.
[0218] Strength tests of the columnar LASCol solids and the
columnar atelocollagen solid were carried out using a small
tabletop tester EZ TEST device (Force Transducer SM-100N-168,
available from Shimadzu Corporation). A longitudinal axis of each
of the columnar LASCol solid pieces and the columnar atelocollagen
solid piece was aligned vertically. Each of the pieces was set so
that the longitudinal axis was perpendicular to a pressurizing unit
of the small tabletop tester EZ TEST device. Then, a stress-strain
curve of each of the pieces was measured by a conventional method.
Then, from inclinations of the curves, tangent moduli of the
columnar LASCol solids and the columnar atelocollagen solid were
calculated.
[0219] Densities of the columnar LASCol solids and the columnar
atelocollagen solid were measured according to the following
method. That is, with use of a standard digital caliper (Digimatic
caliper CD-10AX (product number) available from Mitutoyo
Corporation), a diameter (mm) and a length (mm) of the columnar
shaped product were measured, and a volume (cm.sup.3) of the
cylinder was calculated from a radius, the length, and the circular
constant. In addition, a weight (mg) of the columnar solid was
measured using a semi-microelectronic analytical balance (LIBROR
AEL-40SM (product number) available from Shimadzu Corporation). The
weight (mg) was divided by the volume (cm.sup.3) to calculate the
density (mg/cm.sup.3).
[0220] Table 1 indicates collagen concentrations of solutions used
in preparing the columnar LASCol solids and the columnar
atelocollagen solid and densities and tangent moduli of the LASCol
solids and the atelocollagen solid after freeze drying.
[0221] From Table 1, it can be seen that the use of LASCol makes it
possible to obtain the LASCol solid having a large density and a
large tangent modulus. In contrast, it can be seen that, when
atelocollagen is used, only an atelocollagen solid having a small
density and a small tangent modulus can be obtained.
TABLE-US-00002 TABLE 1 Before freeze After freeze drying drying
(LASCol solid/ (Collagen atelocollagen solid) solution) Tangent
Concentration Density modulus (mg/mL) (mg/cm.sup.3) (kPa)
Collagen-cysteine 180 264 30,277 protease 150 209 9,465 degradation
product 100 150 304 "LASCol" 50 81 175 30 53 93 Collagen-pepsin 20
26 29 degradation product "Atelocollagen"
Example 6. SEM-EDX Analysis of Ca.sup.2+, Na.sup.+,
Cl.sup.--Impregnated Columnar LASCol Solid
[0222] In a manner similar to that of Example 1 above, LASCol was
freeze-dried. The freeze-dried LASCol was then added to ultrapure
water containing 5 mM of Ca.sup.2+, 4 mM of Na.sup.+, and 15 mM of
Cl.sup.- so that a final collagen concentration became 50 mg/mL.
Solutions thus obtained were then left to stand in a refrigerator
at 0.degree. C. to 10.degree. C. for 3 days to 10 days. During the
above periods, the solutions were gently mixed without being
bubbled, and the freeze-dried LASCol was thus completely
dissolved.
[0223] The solution in which the LASCol was completely dissolved
was put into a columnar template (diameter: 3.5 mm, length: 2 mm to
5 mm) and was freeze-dried in a manner similar to that of Example
1. A LASCol solid was then taken out from the columnar template to
obtain a Ca.sup.2+, Na.sup.+, Cl.sup.--impregnated columnar LASCol
solid.
[0224] As a comparative example, a Ca.sup.2+, Na.sup.+,
Cl.sup.--non-impregnated columnar LASCol solid was prepared in a
similar manner, except that ultrapure water which did not contain
Ca.sup.2+, Na.sup.+, and Cl.sup.- was used instead of the ultrapure
water containing 5 mM of Ca.sup.2+, 4 mM of Na.sup.+, and 15 mM of
Cl.sup.-.
[0225] Then, cut surfaces of the Ca.sup.2+, Na.sup.+,
Cl.sup.--impregnated columnar LASCol solid and cut surfaces of the
Ca.sup.2+, Na.sup.+, Cl.sup.--non-impregnated columnar LASCol solid
were imaged by a scanning electron microscope (SU3500 available
from Hitachi High-Technologies Corporation) (acceleration voltage:
15 kV, spot intensity: 60) in a manner similar to that of Example
2. In addition, a scanning electron microscope/energy dispersive
x-ray spectroscope (SEM-EDX, OCTANE PRIME (model number) available
from EDAX Japan) was used to detect element ions contained in the
Ca.sup.2+, Na.sup.+, Cl.sup.--impregnated columnar LASCol solid and
the Ca.sup.2+, Na.sup.+, Cl.sup.--non-impregnated columnar LASCol
solid. The results are shown in (a) through (d) of FIG. 6 and Table
2.
[0226] (a) of FIG. 6 shows a cross-sectional image along a
longitudinal axis of the Ca.sup.2+, Na.sup.+,
Cl.sup.--non-impregnated columnar LASCol solid. (b) of FIG. 6 shows
a result of SEM-EDX analysis of the cross section along the
longitudinal axis of the Ca.sup.2+, Na.sup.+,
Cl.sup.--non-impregnated columnar LASCol solid. (c) of FIG. 6 shows
a cross-sectional image along a lateral axis of the Ca.sup.2+,
Na.sup.+, Cl.sup.--impregnated columnar LASCol solid. (d) of FIG. 6
shows a result of SEM-EDX analysis of the cross section (in
particular, an area surrounded by the square in (c) of FIG. 6)
along the lateral axis of the Ca.sup.2+, Na.sup.+,
Cl.sup.--impregnated columnar LASCol solid. Table 2 shows data
obtained by quantifying amounts of element ions detected in (d) of
FIG. 6. The LASCol solid was proved to contain all the elements of
Na, Cl, and Ca.
TABLE-US-00003 TABLE 2 Signal intensity NaK PtM ClK CaK Value 2.52
20.48 9.21 3.23
[0227] (a) through (d) of FIG. 6 and Table 2 reveal that a
plurality of various substance components, notably Ca.sup.2+,
Na.sup.+, Cl.sup.-, can be contained in the LASCol solid in
accordance with an embodiment of the present invention at any
collagen concentration. In other words, by mixing ultrapure water
with optional substance components with which the solid is to be
impregnated prior to freeze drying, a substance-impregnated
collagen solid can be easily prepared.
Example 7. SEM-EDX Analysis-2 of Ca.sup.2+-Impregnated Columnar
LASCol Solid
[0228] A LASCol solid was prepared in a manner similar to that of
Example 1, except that ultrapure water was added so that a
concentration of LASCol became 50 mg/mL instead of 100 mg. After
freeze drying, the LASCol solid was taken out from a columnar
template (diameter: 3.5 mm, length: 10 mm) to obtain a columnar
LASCol solid. The LASCol solid was immersed in ultrapure water
which contained 10 mM of Ca.sup.2+ and was kept at 37.degree. C.
for 15 minutes while keeping warm, and then the LASCol solid was
freeze-dried again to prepare a Ca.sup.2+-impregnated columnar
LASCol solid.
[0229] The Ca.sup.2+-impregnated columnar LASCol solid thus
obtained was observed by scanning electron microscopy and analyzed
by SEM-EDX in a manner similar to that of Example 6. The results
are shown in (e) and (f) of FIG. 6 and Table 3. (e) of FIG. 6 shows
an image of an outer surface of the Ca.sup.2+-impregnated columnar
LASCol solid. (f) of FIG. 6 shows a result of SEM-EDX analysis of
the outer surface of the Ca.sup.2+-impregnated columnar LASCol
solid. Table 3 shows data obtained by quantifying amounts of
element ions detected in (f) of FIG. 6.
TABLE-US-00004 TABLE 3 Signal intensity CK NK OK CaK Value 132.44
4.69 33.73 8.01
[0230] (e) and (f) of FIG. 6 and Table 3 reveal that any of various
substances, notably Ca.sup.2+, can be contained in the LASCol solid
in accordance with an embodiment of the present invention. That is,
it was possible to prepare the LASCol solid containing an intended
substance component by taking out a LASCol solid having an
arbitrary shape and an arbitrary density after freeze drying, and
then immersing the LASCol solid in an appropriate solution in which
the intended substance component with which the sold was to be
impregnated was dissolved at an arbitrary concentration. The
compound-impregnated LASCol solid in accordance with an embodiment
of the present invention does not change the structure and
properties of LASCol contained in the solid, and therefore the
compound-impregnated LASCol solid can be used even in vivo.
Moreover, in Example 7, the LASCol solid is not dissolved in the
solution containing the optional substance component with which the
solid is to be impregnated. Therefore, change in solubility of the
substance component with respect to the solution due to dissolution
of the LASCol solid in the solution can be ignored. Furthermore, a
LASCol solid containing a plurality of intended substance
components can be prepared as follows: one of the plurality of
substance components is dissolved simultaneously with LASCol in the
same solution, and a mixture thus obtained is freeze-dried; and
then a LASCol solid containing the substance component is immersed
in an appropriate solution in which the other substance component
has been dissolved. In this Example, element ions contained in the
LASCol solid in accordance with the present invention were
quantified without destroying the LASCol solid. Note that, in the
present invention, the substance with which the solid is to be
impregnated is not particularly limited and can be any
substance.
Example 8. Columnar LASCol Solid Having Small Holes
[0231] A columnar LASCol solid was prepared in a manner similar to
that of Example 1, except that 100 mg of the freeze-dried LASCol
described above was added to ultrapure water so that a collagen
concentration became 100 mg/mL, and the above columnar template
(diameter: 2.5 mm to 3.0 mm, length: 4 mm to 7 mm) was changed to
another columnar template (diameter: 3.5 mm, length: 2 mm to 5 mm).
Three through holes were formed in the obtained columnar LASCol
solid using a needle bar (outer diameter: 200 .mu.m) to obtain an
observation sample. The observation sample was imaged with a
stereoscopic microscope (SZ61, available from Olympus Corporation).
The results are shown in FIG. 7.
[0232] (a) of FIG. 7 is an image of the columnar LASCol solid taken
with top illumination. (b) of FIG. 7 is an image of the columnar
LASCol solid taken with bottom illumination. The three through
holes were maintained with little deformation. In contrast, the
columnar atelocollagen solid (diameter: 3.5 mm, length: 2 mm to 5
mm) which was prepared in a manner similar to that of Example 1 and
had a collagen concentration of 20 mg/mL could not have through
holes (not shown). This is because the columnar atelocollagen solid
has low mechanical strength and many voids and therefore, even
though holes are formed, shapes of the holes cannot be kept
unchanged or the holes are closed. The collagen solid in accordance
with an embodiment of the present invention can have small holes in
any size and in any number.
Example 9. SEM-EDX Analysis-3 of Ca.sup.2+-Impregnated Columnar
LASCol Solid
[0233] Ca.sup.2+-impregnated columnar LASCol solids having
different molarities of Ca.sup.2+ were obtained by taking out
freeze-dried LASCol solids from columnar templates (diameter: 3.5
mm, length: 5 mm to 10 mm) in a manner similar to that of Example
6, except that 100 mg of LASCol was added to ultrapure water
containing 10 mM of Ca.sup.2+ or 20 mM of Ca.sup.2+ so that a final
collagen concentration became 50 mg/mL, and 100 mg of LASCol was
added to ultrapure water containing 5 mM of Ca.sup.2+ or 10 mM of
Ca.sup.2+ so that a final collagen concentration became 100
mg/mL.
[0234] Then, insides of the Ca.sup.2+-impregnated columnar LASCol
solids having different molarities of Ca.sup.2+ were imaged by a
scanning electron microscope (SU3500 available from Hitachi
High-Technologies Corporation) (acceleration voltage: 15 kV, spot
intensity: 60) in a manner similar to that of Example 2. In
addition, a scanning electron microscope/energy dispersive x-ray
spectroscope (SEM-EDX, OCTANE PRIME (model number) available from
EDAX Japan) was used to detect element ions contained in the
Ca.sup.2+-impregnated columnar LASCol solids having different
molarities of Ca.sup.2+. The results are shown in (a) through (h)
of FIG. 8 and Table 4.
[0235] (a) of FIG. 8 shows an image of the 10 mM
Ca.sup.2+-impregnated columnar LASCol solid (final collagen
concentration: 50 mg/mL). (b) of FIG. 8 shows the result of SEM-EDX
analysis in a whole screen area of (a) of FIG. 8. (c) of FIG. 8
shows an image of the 20 mM Ca.sup.2+-impregnated columnar LASCol
solid (final collagen concentration: 50 mg/mL). (d) of FIG. 8 shows
the result of SEM-EDX analysis in a whole screen area of (c) of
FIG. 8. (e) of FIG. 8 shows an image of the 5 mM
Ca.sup.2+-impregnated columnar LASCol solid (final collagen
concentration: 100 mg/mL). (f) of FIG. 8 shows the result of
SEM-EDX analysis in a whole screen area of (e) of FIG. 8. (g) of
FIG. 8 shows an image of the 10 mM Ca.sup.2+-impregnated columnar
LASCol solid (final collagen concentration: 100 mg/mL). (h) of FIG.
8 shows the result of SEM-EDX analysis in a whole screen area of
(g) of FIG. 8.
[0236] Table 4 shows data obtained by quantifying intensity of
element ions detected in FIG. 8. In the LASCol solid prepared using
the degradation product having a collagen concentration of 50
mg/mL, CaK/NK is a numerical value calculated by setting the
intensity of N (NK) as a denominator and the intensity of Ca (CaK)
as a numerator. Assuming that a value of CaK/NK was 1 when 10 mM of
Ca.sup.2+ was used, a value of CaK/NK was 2.0 when 20 mM of
Ca.sup.2+ was used. In addition, in the LASCol solid prepared using
the degradation product having a collagen concentration of 100
mg/mL, CaK/NK was similarly calculated. Assuming that a value of
CaK/NK was 1 when 5 mM of Ca.sup.2+ was used, a value of CaK/NK was
1.8 when 10 mM of Ca.sup.2+ was used. That is, when the molarity of
Ca.sup.2+ with which the solid was impregnated in the solvent was
doubled, the value of CaK/NK of the Ca.sup.2+-impregnated columnar
LASCol solid was almost doubled. In other words, it has been found
that the intensity (amount) of Ca detected in the LASCol solid was
increased in proportion to the molar ratio of an optional substance
(e.g., Ca.sup.2+) in ultrapure water containing the optional
substance (e.g., Ca.sup.2+) used in preparing the
Ca.sup.2+-impregnated columnar LASCol solid. Therefore, it was
proved that the Ca.sup.2+-impregnated columnar LASCol solid
contained Ca in an amount depending on the molarity of Ca.sup.2+
contained in the ultrapure water when the LASCol solid was
prepared.
TABLE-US-00005 Tabie 4 50 mg/mL LASCol 100 mg/mL LASCol Signal 10
mM 20 mM 5 mM 10 mM intensity Ca.sup.2+ Ca.sup.2+ Ca.sup.2+
Ca.sup.2+ NK 10.33 11.29 14.51 12.73 CaK 4.12 9.21 1.83 2.81 CaK/NK
0.399 0.816 0.126 0.221 CaK/NK ratio 1 2 -- -- -- -- 1 1.8
[0237] From (a) through (h) of FIG. 8 and Table 4, it has been
found that the LASCol solid in accordance with an embodiment of the
present invention can contain Ca.sup.2+ while increasing or
decreasing an intended amount of Ca.sup.2+. In other words, by
mixing, at any concentration, ultrapure water with an optional
substance component with which the solid is to be impregnated prior
to freeze drying, a collagen solid can be easily prepared which is
impregnated with the intended substance component in a necessary
amount.
Example 10. Evaluation of Bone Regenerative Ability in Animal
Experiment
[0238] [10-1. Preparation of Columnar LASCol Solid for
Implantation]
[0239] A solution containing LASCol was sterilized by filtration
and freeze drying before being formed into a columnar shape. Then,
columnar LASCol solids were prepared by freeze-drying solutions
containing the LASCol after sterilization at predetermined collagen
concentrations (50 mg/mL, 100 mg/mL, and 150 mg/mL) in a manner
similar to that of Example 5 above. A shape of each of the columnar
LASCol solids was the same as a shape of a femur having a 1 mm of
defect, which will be described later. Then, the columnar LASCol
solids were used in Examples described later.
[0240] [10-2. Animal]
[0241] In this Example, rats were used. All rats used were kept in
a 12-hour light-dark cycles at 25.degree. C. and in a pathogen-free
state and were allowed free access to feed and water. All animal
experiments were conducted according to the experimental guidelines
of Kobe University School of Medicine.
[0242] [10-3. Preparation of Femur Defect Rat Model]
[0243] After general anesthesia of the rat and exposure of a femur,
two threaded K-wires were inserted into a proximal site and a
distal site of the femur, respectively, and the K-wires were
connected to each other by an external fixator. Subsequently, a
bone was resected by a width of 1 mm using a small bone saw in the
middle of the shaft of femur to prepare a femur 1 mm defect rat
model. Columnar LASCol solids obtained by freeze-drying solutions
containing the predetermined concentrations (50 mg/mL, 100 mg/mL,
and 150 mg/mL) of LASCol were then implanted into the femur 1 mm
defect rat models, respectively, followed by suturing of the
surgical sites. After the rats were kept for a predetermined period
of time, the bone regeneration status was evaluated.
[0244] Note that 11 cases (n=11) of populations were prepared in
each of which the columnar LASCol solids were not implanted (herein
referred to as "1 mm femur defect population", i.e., control). 21
cases (n=21) of populations were prepared in each of which columnar
LASCol solids obtained by freeze-drying solutions containing LASCol
at a collagen concentration of 50 mg/mL were implanted (herein
referred to as "50 mg/mL LASCol solid implanted population"). 4
cases (n=4) of populations were prepared in each of which columnar
LASCol solids obtained by freeze-drying solutions containing LASCol
at a collagen concentration of 100 mg/mL were implanted (herein
referred to as "100 mg/mL LASCol solid implanted population"). 7
cases (n=7) of populations were prepared in each of which columnar
LASCol solids obtained by freeze-drying solutions containing LASCol
at a collagen concentration of 150 mg/mL were implanted (herein
referred to as "150 mg/mL LASCol solid implanted population").
[0245] (a) of FIG. 9 shows an image of preparing a 4 mm femur
defect rat model by inserting threaded K-wires. (b) of FIG. 9 shows
an image of implanting the columnar LASCol solid into the 4 mm
femur defect rat model.
Example 11. Evaluation with Medical Imaging Technology
[0246] [11-1. Evaluation Criteria Based on Modified RUST Score]
[0247] Evaluation of a degree of bone adhesion based on
radiographic images (which will be described later) was conducted
in accordance with modified RUST scores. Evaluation criteria in
which the evaluation criteria of radiographic union scale in tibial
(RUST) fracture score described in [Whelan D. B. et al. The Journal
of Trauma, 2010] were partially modified were used in this test
(see FIG. 10). In this test, five-level evaluation criteria, i.e.,
score 1 through score 5 were used. In those scores, the higher
number indicates that better bone adhesion is in progress. In this
Example, it was determined that "bone adhesion" occurred when the
modified RUST score based on the radiographic image was evaluated
to be the score 4 or 5.
[0248] [11-2. Radiographic Image Evaluation]
[0249] Immediately after, 14 days after, and 28 days after
implantation of the columnar LASCol solids, the populations were
imaged using a radiography device (Qpix VPX-30E available from
TOSHIBA).
[0250] FIG. 11 shows radiographic images of the 1 mm femur defect
population and the LASCol solid implanted populations taken
immediately after implantation, 14 days after implantation, and 28
days after implantation.
[0251] Table 5 shows the number of individuals in which bone
adhesion occurred and a calculation result of a ratio of
individuals in which bone adhesion occurred in each of the
populations on the 28th day after implantation.
TABLE-US-00006 TABLE 5 Population in which columnar LASCol solids
were implanted into 1 mm femur defect rat models 50 mg/mL 100 mg/mL
150 mg/mL 1 mm LASCol LASCol LASCol femur defect solid solid solid
population implanted implanted implanted (Control) population
population population Number of 2 (11) 11 (21) 4 (4) 7 (7) bone
adhesion individuals (n) Bone adhesion 18% 52% 100% 100% ratio
[0252] As shown in FIG. 11 and Table 5, in the 1 mm femur defect
population, 2 of 11 cases had bone adhesion, and the ratio of bone
adhesion was 18%. In the 50 mg/mL LASCol solid implanted
population, 11 of 21 cases had bone adhesion, and the ratio of bone
adhesion was 52%. In the 100 mg/mL LASCol solid implanted
population, 4 of 4 cases had bone adhesion, and the ratio of bone
adhesion was 100%. In the 150 mg/mL LASCol solid implanted
population, 7 of 7 cases had bone adhesion, and the ratio of bone
adhesion was 100%. That is, the 50 mg/mL, 100 mg/mL, and 150 mg/mL
LASCol solid implanted populations showed that bone adhesion was in
progress on the 28th day after implantation.
[0253] [11-3. Bone Adhesion Evaluation Based on Modified RUST
Score]
[0254] The radiographic images of the 1 mm femur defect population
and the LASCol solid implanted populations taken 28 days after
implantation were evaluated for bone adhesion based on the modified
RUST score. Average scores of the respective populations were
calculated and are shown in FIG. 12. The scores of the respective
populations were also statistically evaluated (p=0.01).
[0255] As shown in FIG. 12, in the 1 mm femur defect population,
the modified RUST score was approximately 2. In contrast, the 50
mg/mL, 100 mg/mL, and 150 mg/mL LASCol solid implanted populations
had higher modified RUST scores than that of the 1 mm femur defect
population. In addition, the modified RUST scores were higher in
the LASCol solid implanted populations of 100 mg/mL and 150 mg/mL,
as compared with the 50 mg/mL LASCol solid implanted
population.
[0256] [11-4. .mu.CT Image Evaluation]
[0257] 14 days and 28 days after implantation, computed tomography
(CT) was carried out with a .mu.CT device (R_mCT; Rigaku
Mechatronics Co., Ltd., Tokyo, Japan) to evaluate a degree of
progress of bone adhesion. Rats were euthanized by cervical
dislocation and subjected to computed tomography, followed by
histological evaluation as described later. The results are shown
in FIGS. 13 through 17.
[0258] FIG. 13 shows CT images of the 1 mm femur defect population
and the LASCol solid implanted populations taken 28 days after
implantation. As shown in FIG. 13, the LASCol solid implanted
populations showed bone adhesion at any of the LASCol solid
concentrations. In particular, complete bone adhesion was observed
in the LASCol solid implanted populations of 100 mg/mL and 150
mg/mL.
[0259] FIG. 14 shows CT images of the 100 mg/mL LASCol solid
implanted population taken 28 days after implantation. A through D
represent four individuals, respectively. As shown in FIG. 14,
complete bone adhesion was observed in all rats.
[0260] FIG. 15 shows CT images of the 150 mg/mL LASCol solid
implanted population taken 14 days after implantation. A through D
show images of representative four individuals of the seven
individuals (n=7). It can be seen that bone tissue formation is in
progress and bone regeneration is in progress particularly at the
sites indicated by the arrows in the images.
[0261] FIG. 16 shows CT images of the 150 mg/mL LASCol solid
implanted population taken 28 days after implantation. A through D
show images of representative four individuals of the seven
individuals (n=7). It was seen that bone tissue formation
progressed to achieve complete bone adhesion particularly at the
sites indicated by the arrows in the images, as compared with the
case on the 14th day after implantation (shown in FIG. 15).
Example 12. Histological Evaluation
[0262] [12-1. Femur Extraction]
[0263] The femurs were extracted, 14 days and 28 days after
implantation, from the 50 mg/mL, 100 mg/mL and the 150 mg/mL LASCol
solid implanted populations which had been subjected to [11-4.
.mu.CT image evaluation] above. FIG. 17 shows an image of a femur
extracted from a rat in the 150 mg/mL LASCol solid implanted
population taken 28 days after implantation.
[0264] As shown in FIG. 17, bone adhesion was observed 28 days
after implantation at the site where the 150 mg/mL LASCol solid was
implanted in a rat having the 1 mm defect of femur.
[0265] [12-2. Evaluation Criteria Based on Allen's Score]
[0266] In the histological evaluation, the degree of progress of
bone adhesion in stained images (described later) was evaluated in
accordance with Allen's scores. The evaluation criteria were in
accordance with the evaluation criteria described in [H. L. Allen
et al. Acta Orthopaedica Scandinavica, 1980]. The evaluation
criteria of the degree of progress of bone adhesion (herein
referred to as "Allen's score") are shown in FIG. 18.
[0267] [12-3. HE Stain]
[0268] A sectioned tissue of the femur extracted in [12-1. Femur
extraction] above was prepared, and hematoxylin eosin stain (HE
stain) was carried out on that sectioned tissue. In the HE stain,
hematoxylin used was Mayer's Hematoxylin (product number: 30002,
available from MUTO PURE CHEMICALS CO., LTD.) and eosin used was
Eosin Y (product number: 058-00062, available from Wako), which
were used in accordance with the respective use methods to stain
the sectioned tissue. The sectioned tissue after staining was
observed with an optical microscope (product name: BA-X700,
available from Keyence Corporation, Osaka, Japan).
[0269] FIG. 19 shows images of the HE stained sectioned femur
tissues of the 50 mg/mL LASCol solid implanted population and the
HE stained sectioned femur tissues of the 1 mm femur defect
population (control), which were taken 14 days and 28 days after
implantation. As shown in FIG. 19, it is seen that bone adhesion
was in progress on the 14th day after implantation in the femur of
the 50 mg/mL LASCol solid implanted population, and bone adhesion
was advancing on the 28th day after implantation from the state on
the 14th day after implantation.
[0270] [12-4. SO Stain]
[0271] With respect to the sectioned tissue used in [12-3. HE
stain]
[0272] above, safranin O stain (SO stain) was carried out. In the
SO stain, safranin O used was Fastgreen FCF (product number: 10720,
available from CHROMA-GESELLSCHAFT) and oil red used was Basic Red2
(product number: GB01-PALO, available from Tokyo Chemical Industry
Co., Ltd.), which were used in accordance with the respective use
methods to stain the sectioned tissues. The sectioned tissues after
staining were observed with an optical microscope (product name:
BA-X700, available from Keyence Corporation, Osaka, Japan). The
results are shown in FIGS. 20 and 22 through 24.
[0273] FIG. 20 shows images of the SO stained sectioned femur
tissues of the 50 mg/mL LASCol solid implanted individuals and the
SO stained sectioned femur tissues of the 1 mm femur defect
individuals (control), which were taken 14 days and 28 days after
implantation. As shown in FIG. 20, in any of the sectioned femur
tissues on the 14th day after implantation, formation of cartilage
tissue was observed at orange-stained sites (indicated by the
arrows in FIG. 20). As a result of evaluation based on the Allen's
score, the evaluation scores were all Grade 2. In the 50 mg/mL
LASCol solid implanted individual on the 28th day after
implantation, bone adhesion progressed and the evaluation based on
the Allen's score was Grade 4. In contrast, cartilage tissues
(indicated by the arrows in FIG. 20) were also observed in the
control 28 days after implantation but the evaluation result based
on the Allen's score was Grade 3. That is, bone adhesion was
incomplete.
[0274] FIG. 21 shows images of sectioned femur tissues of four
individuals out of the 100 mg/mL LASCol solid implanted population
on the 28th day after implantation. As shown in FIG. 21, in the 100
mg/mL LASCol solid implanted population, no cartilage tissue was
observed in all the individuals, and the evaluation results based
on the Allen's score were all Grade 4. That is, it was found that
implanting the 100 mg/mL LASCol solid into the rat resulted in
complete bone adhesion on the 28th day after implantation.
[0275] FIG. 22 shows images of sectioned femurs of four individuals
out of the 150 mg/mL LASCol solid implanted population on the 28th
day after implantation. As shown in FIG. 22, in the 150 mg/mL
LASCol solid implanted population, the evaluation results based on
the Allen's score were Grade 4 in three individuals out of four
individuals (i.e., cartilage tissue was observed in one
individual). That is, it was found that implanting the 150 mg/mL
LASCol solid into the rat significantly promoted progress of bone
adhesion on the 28th day after implantation.
[0276] Further, FIG. 23 shows results of carrying out the above SO
stain and evaluation based on the Allen's score 28 days after
implantation with respect to: 10 individuals (n=10) of the 50 mg/mL
LASCol solid implanted population; four individuals (n=4) of the
100 mg/mL LASCol solid implanted population; four individuals (n=4)
of the 150 mg/mL LASCol solid implanted population, and seven
individuals (control, n=7) of the 1 mm femur defect population. The
scores for the LASCol solid implanted populations of respective
concentrations and the 1 mm femur defect population were then
statistically evaluated. As shown in FIG. 23, on the 28th day after
implantation, the 50 mg/mL LASCol solid implanted population was
found to have the higher Allen's score than the control population,
and have the tendency of progressed bone adhesion (p=0.06).
Moreover, the 100 mg/mL LASCol solid implanted population and the
150 mg/mL LASCol solid implanted population had significantly
higher Allen's scores and had progressed bone regeneration, as
compared with the control population (p<0.05).
Example 13. CaCO.sub.3-Containing LASCol Solid
[0277] Elements constituting hard tissues such as calcium and
phosphorus have a function of promoting bone formation. Therefore,
the inventors have considered that, if a bone prosthetic material
for sustained release of calcium ions is prepared, the function of
promoting bone formation can be expected. Specifically, the
inventors thought as follows: by incorporating calcium carbonate as
a calcium source into the LASCol solid, sustained release of
calcium ions in vivo and further enhancement of osteoinductivity
could be expected. Under this hypothesis, the following test was
conducted.
[0278] A columnar LASCol solid was prepared in a manner similar to
that of Example 6, except that the final collagen concentration was
150 mg/mL and a solution containing 10 mM of calcium carbonate
(CaCo.sub.3) was used. As a template, a columnar template
(diameter: 3.5 mm, length: 1 mm) was used. Here, the obtained
LASCol solid is referred to as "CaCo.sub.3-impregnated 150 mg/mL
LASCol solid".
[0279] A 1 mm femur defect population was prepared in a manner
similar to that of Example 10, except that a femur was resected by
a width of 1 mm in each rat. Four individuals (n=4) of the
CaCo.sub.3-impregnated 150 mg/mL LASCol solid implanted population
were prepared. After the rats were kept for a predetermined period
of time, the bone regeneration status was evaluated.
[0280] 14 days after implantation, computed tomography was carried
out with a .mu.CT device in a manner similar to that in [11-4.
.mu.CT image evaluation] to evaluate a degree of progress of bone
adhesion. The result is shown in FIG. 24.
[0281] FIG. 24 shows a typical CT image in the
CaCo.sub.3-impregnated 150 mg/mL LASCol solid implanted population
on the 14th day after implantation. As shown in FIG. 24, vigorous
bone tissue formation was in progress on the 14th day after
implantation. For example, in FIG. 24, it can be seen that bone
regeneration is clearly promoted, as compared with FIG. 15
described above.
[0282] Based on those results, the CaCO.sub.3-containing LASCol
solid is expected to be clinically applied as an innovative bone
prosthetic material for sustained release of calcium ions in a bone
defect site, in addition to its osteoinductivity.
Example 14, bFGD-Containing LASCol Solid
[0283] Fibroblast growth factors (hereinafter referred to as
"bFGF") are known to promote bone regeneration by proliferating and
differentiating undifferentiated mesenchymal stem cells. The LASCol
solid begins to be degraded when the LASCol solid is embedded in a
living body. Based on this fact, the inventors considered that the
LASCol solid has a function as a bone prosthetic material which
serves as a sustained release material which continues to release a
physiologically active substance such as bFGF. Based on this idea,
the inventors prepared a LASCol solid containing recombinant human
basic fibroblast growth factors (product name: Fiblast, KAKEN
PHARMACEUTICAL CO., LTD.) An effect of the LASCol solid containing
basic fibroblast growth factors on bone regeneration was
investigated using a critical femur deficiency rat model, which is
generally considered not to show bone adhesion in the natural
course.
[0284] A columnar LASCol solid was prepared in a manner similar to
that of Example 6, except that a final collagen concentration was
100 mg/mL and a solution containing 12 .mu.g of fibroblast growth
factors (bFGF, product name: Fiblast, available from KAKEN
PHARMACEUTICAL CO., LTD) was used. As a template, a columnar
template (diameter: 3.5 mm, length: 4 mm) was used. Here, the
obtained LASCol solid is referred to as "bFGF-impregnated 100 mg/mL
LASCol solid".
[0285] A 4 mm femur defect population was prepared in a manner
similar to that of Example 10. One individual (n=1) was prepared as
a control (in which nothing was implanted into the bone defect
site) and one individual (n=1) was prepared as the bFGF-impregnated
100 mg/mL LASCol solid implanted individual. After the rats were
kept for a predetermined period of time, the bone regeneration
status was evaluated.
[0286] 35 days after implantation, computed tomography was carried
out with a .mu.CT device in a manner similar to that in [11-4.
.mu.CT image evaluation] to evaluate a degree of progress of bone
adhesion. The results are shown in FIG. 25.
[0287] FIG. 25 shows CT images of (a) the bFGF-impregnated 100
mg/mL LASCol solid implanted individual and (b) the 4 mm femur
defect individual (control) taken 35 days after implantation. As
shown in FIG. 25, bone regeneration was seen in the
bFGF-impregnated 100 mg/mL LASCol solid implanted individual,
whereas bone regeneration was not seen in the 4 mm femur defect
individual.
[0288] As such, the results shown in FIGS. 24 and 25 revealed that
the CaCO.sub.3-impregnated LASCol solid and the bFGF-impregnated
LASCol solid are expected to be clinically applied as a novel bone
regeneration material that does not require cell
transplantation.
INDUSTRIAL APPLICABILITY
[0289] The present invention can be widely utilized in the field of
materials (e.g., in the field of bone disease treatment, or in the
field of cell culture). More specifically, the present invention
can be widely used in treatment of fractures, bone tumors, and
osteomyelitis, or in in vitro cell culture.
Sequence CWU 1
1
3110PRTArtificial SequencePartial Sequence of
Proteinmisc_feature(2)..(3)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(6)Xaa can be any naturally occurring amino
acidmisc_feature(8)..(9)Xaa can be any naturally occurring amino
acid 1Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly1 5 10222PRTArtificial
SequencePartial Sequence of Proteinmisc_feature(2)..(3)Xaa can be
any naturally occurring amino acidmisc_feature(5)..(6)Xaa can be
any naturally occurring amino acidmisc_feature(8)..(9)Xaa can be
any naturally occurring amino acidmisc_feature(11)..(12)Xaa can be
any naturally occurring amino acidmisc_feature(14)..(15)Xaa can be
any naturally occurring amino acidmisc_feature(17)..(18)Xaa can be
any naturally occurring amino acidmisc_feature(20)..(21)Xaa can be
any naturally occurring amino acid 2Gly Xaa Xaa Gly Xaa Xaa Gly Xaa
Xaa Gly Xaa Xaa Gly Xaa Xaa Gly1 5 10 15Xaa Xaa Gly Xaa Xaa Gly
20313PRTArtificial SequencePartial Sequence of
Proteinmisc_feature(1)..(3)Xaa can be any naturally occurring amino
acidmisc_feature(5)..(6)Xaa can be any naturally occurring amino
acidmisc_feature(8)..(9)Xaa can be any naturally occurring amino
acidmisc_feature(11)..(12)Xaa can be any naturally occurring amino
acid 3Xaa Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly Xaa Xaa Gly1 5 10
* * * * *